Molecular Human Reproduction, Vol. 8, No. 12, 1087-1095,
December 2002
© 2002 European Society of Human Reproduction and Embryology
Regulation of ovarian function |
Isolation, characterization and expression of the human Factor In the Germline alpha (FIGLA) gene in ovarian follicles and oocytes
1 Academic Unit of Paediatrics, Obstetrics and Gynaecology, University of Leeds, D Floor, Clarendon Wing, Leeds General Infirmary, Belmont Grove, Leeds LS2 9NS, 2 The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, VA 23507-1627, USA and 3 Assisted Conception Unit, Clarendon Wing, Leeds General Infirmary, Belmont Grove, Leeds LS2 9NS, UK
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Abstract |
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The Factor In the Germline alpha (FIG
) transcription factor regulates expression of the zona pellucida proteins ZP1, ZP2 and ZP3 and is essential for folliculogenesis in the mouse. Using the published mouse Figla sequence, BLAST searches identified a human chromosome 2 BAC clone with high sequence identity. Using PCR primers derived from this clone, amplicons derived from ovarian follicles and mature oocytes revealed 100% identity with the appropriate human BAC clone, the expected homology with the mouse Figla gene sequence, and homology on translation with the FIG protein identified in the Japanese rice fish, medaka (Oryzias latipes). PCR expression profiling of this transcript revealed FIGLA mRNA expression in cDNA derived from ovarian follicles (5/5 samples from the primordial through to the secondary stage) mature oocytes (6/9 samples), and less frequently in preimplantation embryos (2/7 samples). Subsequent BLAST searches revealed the predicted full length coding sequence of the human FIG protein which demonstrates 68 and 25% similarity overall to mouse and medaka proteins respectively, with 96 and 57% identity respectively within the basic helix–loop–helix region. This confirms our identification of the human homologue for this gene which maps to chromosome 2p12. Further work is required to understand its role in normal human oocyte development and the potential involvement in human infertility.
FIG transcription factor/FIGa/FIGLA/oocytes/ovarian follicles/zona pellucida
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Introduction |
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TopAbstractIntroductionResultsDiscussionAcknowledgementsReferences |
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The growth and development of mammalian oocytes requires, amongst other things, a co-ordinated programme of gene expression events that occur in developing follicles. The mammalian oocyte itself regulates the development of ovarian follicles (Eppig et al., 2002
) suggesting that genes that are expressed early on, and perhaps specifically during oocyte growth and development, are likely to play important roles in this process. To date, a number of growth factors such as growth differentiation factor-9, bone morphogenetic protein 15 (Dong et al., 1996; Dube et al., 1998) and also proteins expressed on the cell surface, for example KIT receptor (Manova et al., 1993), have been found to contribute to folliculogenesis. Key roles during folliculogenesis and oogenesis are also suggested for the OCT4 transcription factor, a marker of totipotency which is expressed in mature human oocytes and preimplantation embryos (Abdel-Rahmen et al., 1995), primordial germ cells, and the female germ line (Rosner et al., 1990). Anti-Müllerian hormone (AMH) is another key regulator of early follicle growth and determines the sensitivity of ovarian follicles to FSH (Durlinger et al., 2001).
Studies of murine ovarian folliculogenesis have revealed that a basic helix–loop–helix (bHLH) transcription factor, Factor In the Germline alpha (FIG), plays a crucial role in the formation of primordial follicles (Soyal et al., 2000). Thus, targeted mutagenesis of the murine Figla gene that encodes the FIG transcription factor generates germ cell-depleted ovaries, and homozygous Figla null females are therefore sterile. In contrast, homozygous Figla null male mice appear unaffected and are fertile. The murine FIG transcription factor also plays a role in regulating the coordinate expression of genes that encode the zona pellucida (ZP) glycoproteins ZP1, ZP2 and ZP3. The transcription factor FIG binds to the E-box in the ZP gene promoters in vitro as a heterodimer with the class A bHLH protein E12 to regulate ZP gene expression (Liang et al., 1997). Interestingly, a Figla homologue showing 53% identity that is restricted to the bHLH region, has been discovered during an analysis of genes expressed during early oogenesis in the Japanese rice fish, medaka (Oryzias latipes), suggesting an equivalent role in divergent species (Kanamori, 2000). The critical roles of FIG demonstrated in murine oogenesis, and apparently during early oogenesis in other species, suggest that this factor might be important in human oogenesis, and so warrants the isolation and characterization of the human FIGLA gene.
We have generated cDNA pools from various stages of human folliculogenesis. Utilizing these resources, we report the cloning, characterization and expression analysis in staged human ovarian follicles and mature oocytes, of the human FIGLA homologue. The expression of FIGLA mRNA compared to expression of mRNA of ZP2, ZP3, and the OCT4 transcription factor in human ovarian follicles, granulosa cells, oocytes and preimplantation embryos is discussed.
Materials and methods
Sample collection
Human ovarian follicles
An ovarian cortex biopsy was donated for research under an ethically approved protocol by a 22 year old woman attending Leeds General Infirmary hospital for gynaecological surgery. Following harvest, the tissue was cryopreserved according to a published protocol (Newton et al., 1996). Following rapid thawing, early staged follicles were harvested from the tissue by needle dissection after a brief period of digestion in collagenase 1A and DNase (Oktay et al., 1997a). Isolated follicles were measured and classified using a Nikon Diaphot microscope fitted with Hoffman optics. Follicular developmental stages were classified according to published criteria (Oktay et al., 1997b). Briefly, primordial follicles were indicated by the presence of an oocyte of 34–38 µm surrounded by flattened pre-granulosa cells; early primary follicles were represented by an oocyte of 34–53 µm diameter enclosed within a mixture of flattened pre-granulosa cells and cuboidal granulosa cells. Primary follicles were between 52.8–62.4 µm in diameter and were surrounded by a complete layer of cuboidal granulosa cells. Secondary follicles were 62–86 µm in diameter and enclosed by more than one layer of cuboidal granulosa cells. All follicles were photographed for later reference. Follicles with similar sizes or of the same developmental stage were pooled in a microdrop of Dulbecco’s phosphate-buffered saline without calcium or magnesium to avoid adherence. Pooled follicles were then added to 50 µl DynalTM lysis buffer (Dynal, UK) on ice. Follicle-free stromal tissue as assessed by inverted microscope was also collected from the ovarian tissue for comparison.
IVF samples
Human oocytes, which were at the germinal vesicle stage at the time of ICSI, and preimplantation embryos which were surplus to requirement for clinical treatments were collected. Tissue was obtained from 12 patients attending Leeds General Infirmary Assisted Conception Unit for gynaecological surgery and was obtained after informed consent under ethically approved protocols (where appropriate) which were licensed by the Human Fertilisation and Embryo Authority. Oocyte maturity and the stage of embryo development were recorded. For comparison, non-luteinized granulosa cells from antral follicles of 6–10 mm diameter were collected from three patients during immature oocyte recovery as part of an in-vitro maturation programme. Samples were washed in PBS and lysed in 50 µl Dynal lysis buffer on ice. Either one or two oocytes or preimplantation embryos were used for preparing each cDNA sample as indicated in the figure legends.
RNA extraction
Oocytes, preimplantation embryos, ovarian follicles and granulosa cells were lysed in Dynal lysis buffer at 70°C for 10 min and checked under a microscope for completion of lysis. A total of 15 µl of washed oligo(dT)25 magnetic Dynabeads (Dynal) were added to lysed samples, and binding of mRNA was performed for 30 min at room temperature with agitation. Messenger RNA was separated from other cellular components (including genomic DNA) using a magnetic work station and samples were washed in progressive buffer steps according to the manufacturer’s protocol and finally resuspended in 3 µl of distilled water.
SMART cDNA synthesis
One microlitre each of CDS III/3' primer and Smart IV oligonucleotides (SMART library construction kit; Clontech, Palo Alto, CA, USA) were added to each re-suspended mRNA sample and heated to 70°C for 2 min, followed by cooling on ice. Reverse transcription (RT) was then performed with Powerscript Reverse Transcriptase (Clontech) at 42°C for 1 h.
Total cDNA PCR amplification
Total cDNA was prepared by amplification of 10 µl of each RT reaction mixture with PCR primers (5' PCR primer, CDS III/3' primer) for 35 cycles, according to the manufacturer’s protocol (Clontech) on a Perkin–Elmer GeneAmp 480 Thermal Cycler (Perkin–Elmer, CT, USA), to generate 100 µl of PCR amplified total cDNA per sample. At this stage, 6 µl of each cDNA was analysed by gel electrophoresis to confirm a representative spread of cDNA sizes from each sample (data not shown). PCRs for a number of housekeeping genes; glyceraldehyde-3-phosphate dehydrogenase (GAPD, also known as GAPDH or G3PDH), hypoxanthine phosphoribosyl transferase (HPRT) and ß-actin were also performed to check cDNA quality. Samples that repeatedly failed to amplify here were discarded. Ovarian follicular cDNA samples were further tested for expression of the Anti-Müllerian Hormone type II receptor (data not shown) to confirm the specificity of the isolation protocol.
Human FIGLA PCR
Originally, primers were derived from Homo sapiens BAC clone RP11-504O1 from chromosome 2 encompassing the regions showing high homology with mouse Figla sequence. For amplification of putative transcripts of the human FIGLA gene, the forward (5') primer (FIGLA F1) was designed in the first helix of the helix–loop–helix domain. Two reverse (3') primers were designed, one within the second helix of the helix–loop–helix domain (FIGLA R2) (Figure 1b). The reverse primer (FIGLA R1) was designed within a more 3' conserved GA-rich motif. One microlitre of SMART cDNA was used per 25 µl volume PCR reaction of 32 cycles of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C utilizing Advantage 2 Taq (Clontech) and the associated protocol. Single round PCR were performed with each primer set and a hemi-nested PCR was used to further verify data. Human multiple tissue PCR were performed using the PCR parameters above using cDNA from Multiple Tissue cDNA (MTC–Clontech). All primer sequences and relevant accession numbers, references and positions are described in Table I.
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Other PCR protocols
PCR primers for analysis of expression of ZP2, ZP3, OCT4 transcripts are described in Table I
. Cycling conditions were the same as for the FIGLA PCR. Primers for GAPD were obtained from Clontech MTC panels or SMART cDNA synthesis kits and were utilized according to the manufacturer’s protocol. All PCR experiments were repeated a minimum of three times.
Partial cloning, sequencing and full FIGLA characterization in silico
Potential FIGLA bands generated from ovarian follicular and oocyte cDNA were isolated from agarose gels using a Qiagen kit (Qiagen Ltd, Crawley, UK) and subcloned into Invitrogen Topo TA sequencing vector (Invitrogen, Groningen, The Netherlands). Samples were analysed at the Biomolecular Analysis Facility, University of Leeds. cDNA sequences, obtained in both directions (M13 forward and reverse primers) were confirmed by BLAST searching and further analysed using Gene-Jockey II bioinformatics software (Biosoft, Cambridge, UK). All other Amplicons were confirmed by sequencing.
Following confirmation of the sequence homology between our RT–PCR-directed amplicons and published sequence data, and subsequent expression profiling, the partial FIGLA clone sequence was resubmitted for BLAST search against an updated HGMP chromosome 2 sequencing build (build 30, Hs_2_22340_30_24_1).
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Results |
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Bioinformatics
The mouse Figla sequence (MMU91840) was used to perform a BLAST search of human sequences. Originally, this identified several regions of homology with human BAC clone RP11-504O1 from chromosome 2. Particular homology was identified at the DNA level in the region corresponding to the helix–loop–helix region (87%, 101/115 base pairs), and there was further homology more 5' to this region, including the basic region (80% homology, 144/178 base pairs). Although we were initially unable to identify the most 5' or 3' sequences by homology within this BAC clone, subsequent searches using the mouse Figla sequence (and the putative human FIGLA transcripts described here) identified the electronically predicted full length human FIGLA coding sequence from a more recent submission (accession number XM_065957) relevant to build 30 of human chromosome 2 (XM_22340_30_24_1), contig NI_022184, spanning 13 278 bp (1313522–1300244) and containing the full length gDNA for a human FIGLA-like sequence. Analysis of the predicted intron/exon structure of human FIGLA was performed using the model maker facility (NCBI-BLAST) which predicted 4 exons and a protein of 186 amino acids. Figure 1a
shows the predicted human FIGLA coding and protein sequences, and the sequences of the exon/intron boundaries. The structure of the FIGLA gene (LOC130936) and its position relevant to other genes in the chromosome 2 region 2p12 is shown in Figure 1b.
The human FIG transcription factor peptide sequence XP_065957 was compared with mouse and rice fish (medaka) peptides (Figure 2a). The overall amino acid identity between human and mouse peptides was 68%, whilst 25% identity was observed between human and medaka peptides. The human and mouse proteins were observed to be highly similar within the basic helix–loop–helix region with 96% identity over 52 amino acids, whilst 57% identity was recorded between human and medaka peptides for this region (Figure 2b).
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Initial isolation of FIGLA from human ovarian follicles and mature oocytes
To confirm the expression of transcripts of the predicted human FIGLA gene, primers were designed from the regions of highest homology between mouse and human sequences (the helix–loop–helix region, Figure 2b
) for PCR amplification from SMART-amplified cDNA derived from ovarian follicles and oocytes. Examples of the human ovarian follicles obtained by fine dissection and staged by size and morphology are shown in Figure 3. In deciding our experimental approach towards identifying genes that are important during human oogenesis, we had considered the possibility that certain transcripts may be expressed only transiently during the oocyte growth phase. Therefore, in order to account for this eventuality, alongside the isolation of the exclusively primordial, primary and secondary follicle samples, we also collected intermediate stages comprised of mixtures of: (i) primordial to early primary (34–53 µm) and (ii) early primary to primary follicles (48–57 µm). Five developmentally incremental cDNA samples were therefore generated from ovarian follicles, these being: (i) 28 primordial follicles, (ii) 45 primordial–early primary follicles, (iii) 30 early primary–primary follicles, (iv) seven primary and (v) seven secondary follicles. Amplicons of expected sizes were generated for FIGLA F1/R1 and FIGLA F1/R2 primers and also in hemi-nesting experiments with these primers from cDNA derived from the ovarian follicles and mature metaphase II (MII) IVF oocytes (for mRNA expression profile, see Figures 4a and 4b). The FIGLA amplicons were subcloned and sequenced and F1/R1 primer products demonstrated 98–100% concordance with the human BAC clone sequence RP11-504O1 by BLAST analysis, matching two expressed exonic regions, separated by a 2 kb intron. Resubmission of the partial ovarian follicle and oocyte-derived FIGLA clone sequences for BLAST searching at a later stage identified the predicted full length sequence XM_065957 (98–100% identity), with the expected exon–intron junctions. The FIGLA F1/R1 PCR product therefore spans from the last nucleotide of exon 1, through exon 2, and through the majority of exon 3 of the predicted full length sequence (sequence shown in bold type, Figure 1a).
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Expression analysis of human FIGLA in cDNA derived from ovarian follicles, oocytes and preimplantation embryos
To further characterize the expression of the FIGLA transcript throughout human oogenesis and during preimplantation development, two FIGLA PCR reactions were performed on cDNA generated from human ovarian follicles, mature oocytes and preimplantation embryos. Given that the murine FIG
transcription factor plays a critical role in the regulation of expression of the zona pellucida genes, the analysis of expression of human FIGLA transcripts was compared with that of two human zona pellucida genes (ZP2 and ZP3). Also included in this analysis was amplification of the housekeeping gene, GAPD, and the transcription factor, OCT4.
Visual comparison of FIGLA PCR with the housekeeping gene positive control PCR, GAPD (Figure 4a, panel f) indicates which developmental stages express FIGLA transcripts. The ovarian follicle samples (1–5) each contain cDNA derived from a number of follicles (between 7 and 45) and hence the effects of inter-sample variation of gene expression are eliminated in these pooled samples. However, in the cDNA amplifications derived from a single (or sometimes two) mature oocyte, or preimplantation embryos, a number of individual samples per given stage were assayed to account for the possible variations in gene expression between samples. All cDNAs used for expression analysis here have been previously extensively tested and verified as being positive by PCR throughout the developmental series for the expression of >18 additional positive control genes (data not shown).
PCR with primers FIGLA F1/R2 within the helix–loop–helix region (Figure 4a, panel a) demonstrates expression in all ovarian follicular stages from primordial to secondary follicles (lanes 1–5). These products were again subcloned and sequenced and gave a 100% match with accession XM_065957 and the relevant chromosome 2 BAC clones in all samples and colonies sequenced. The expected sequence match to mouse Figla was also identified, and on translation, the Japanese rice fish (medaka) sequence was identified (as predicted in Figure 2a and b). Although 2/5 MII oocyte cDNA samples were positive for FIGLA expression in the presented gel, the overall frequency of expression was closer to 70% (see below). Of the four blastocysts tested, one was positive for FIGLA expression (1/7 preimplantation embryos positive in this assay).
PCR with primers spanning FIGLA intron 2 (FIGLA F1/R1) also generated the expected 294 bp amplicon in all five ovarian follicle samples (Figure 4a, panel b). The amplicon was again confirmed by sequencing. Two MII oocyte cDNA samples (2/5), one blastocyst sample (1/4) and one 4-cell embryo (1/2) were also positive in this PCR.
Further expression analysis of FIGLA transcripts was performed by PCR in cDNA derived from additional MII oocyte samples (Figure 4b, panel a). Using FIGLA F1/R1 primers, the expected 284 bp amplicon was obtained in all three additional MII oocyte samples (non-injected ICSI and failed fertilization injected ICSI oocytes). Furthermore, hemi-nested PCR analysis using FIGLA F1/R2 primers and 1 µl of amplicons from the first round revealed the expected size second round product of 102 bp (Figure 4b, panel b). The total number of MII oocytes tested that were observed to express FIGLA was therefore 6/9 samples (Figures 4a, 4b, 7 and unpublished data).
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ZP3/ZP2 PCR and the identification of a novel human ZP2 splice variant
ZP3 transcripts were detected in all ovarian follicles and oocytes and also all preimplantation embryo samples (Figure 4a
, panel c). ZP2 transcripts demonstrated a similar pattern of expression (Figure 4a panel d), although only 2/4 of the blastocyst samples generated an amplicon. When ZP2 amplicons were separated on gels for a longer period, an additional higher molecular weight band was observed in a number of samples, including ovarian follicles (primordial, the mixture of primordial through to early primary, and the mixture of early primary through to primary follicles) and 3/5 mature oocytes and the 2-cell sample (Figure 5i). Sequencing of this band identified the product as a ZP2 splice variant, with a 33 nucleotide insertion (Figure 5ii) with coding potential (Figure 5iii).
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OCT4 transcription factor
OCT4 transcripts were only detected in 1/5 ovarian follicle samples (Figure 4a
, panel e), the positive sample being the mixture of early primary and primordial follicles. Transcripts of OCT4 were detected in all mature oocytes and blastocysts.
Expression analysis of human FIGLA in cDNA from multiple human tissues
To deduce whether human FIGLA transcripts were germ cell-specific, PCR was performed using intron-spanning FIGLA F1/R1 primers using normalized cDNA derived from multiple human tissues including ovary and testis (Clontech MTC panels). cDNA was also generated from the ovarian tissue biopsy used for ovarian follicle isolation (presumed to contain all cell types). Primordial follicle cDNA, generated by SMART amplification, were also included as controls. Results were compared with those for the housekeeping gene GAPD (Figure 6). No amplification of human FIGLA was seen in any tissue except the control primordial follicle sample. All samples were positive for GAPD. From this lack of amplification in adult ovarian tissue, it would appear that human FIGLA transcripts are rare and/or not detectable using RT–PCR from adult ovary and other tissues.
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Expression analysis of human FIGLA in cDNA from granulosa cells
The cDNA generated from ovarian follicles are likely to include a contribution of mRNA derived from both the oocytes and the granulosa cells. Therefore, to ascertain whether the FIGLA transcripts are expressed in the oocyte and/or the granulosa cells, we generated SMART cDNA from three non-luteinized mural granulosa cell samples which were isolated from 6–10 mm antral follicles from three consenting patients undergoing IVF treatment. These samples were screened to ensure that they were devoid of oocytes prior to lysis. Results of PCR experiments for the intron 2-spanning FIGLA F1/R1 primers did not detect FIGLA transcripts in these granulosa samples, whereas cDNA from primordial follicles and a MII oocyte were positive in this assay (Figure 7
). All samples were positive for housekeeping genes GAPD and HPRT. |
Discussion |
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Greater understanding of the regulation of early human oogenesis requires analysis of the genes expressed either specifically or predominantly during these stages. The small size and restricted availability of human ovarian follicles, oocytes and preimplantation embryos demands that sensitive molecular techniques are employed to generate the maximum amount of information per retrieval for this purpose. Traditional single cell RT–PCR methods provide opportunities for only a limited number of comparative gene expression analyses and are of limited use in cDNA cloning procedures. We therefore generated cDNA samples from the various stages of human ovarian follicles, mature oocytes and preimplantation development using SMART-mediated cDNA synthesis. This method has been previously used to analyse gene expression in human preimplantation embryos and oocytes (Adjaye et al., 1999
; Holding et al., 2000) and creates re-usable libraries or pools of cDNA from samples as small as a single cell. By comparison with PCR analysis of transcripts for housekeeping genes, it is possible to demonstrate the expression status of a given gene for each developmental stage. We believe that this is the first reported isolation and application of such cDNA libraries from staged early human ovarian follicles.
Using these cDNA samples, we have established an expression profile of a transcript representing a putative human FIGLA homologue that is expressed in primordial, primary and secondary stage ovarian follicles and mature oocytes. In addition, expression of the transcript is derived from the oocyte rather than granulosa cells. Based on the full published FIGLA coding sequence data predicted by automatic computational analysis and the homologous sequences derived by cloning RT–PCR amplicons from our samples, we propose that the expression patterns reported herein truly represent those of the human FIGLA gene. The human gene is comprised of four exons, maps to chromosome 2p12 and encodes a peptide of 186 amino acids. Comparison of human, mouse and medaka FIG proteins reveals that the highest degree of similarity occurs within the basic helix–loop–helix region (bHLH), suggesting evolutionary pressure for the conservation of this region between species. This is likely to be due to common essential functional requirements such as the binding of E-box motifs in the promoters of the zona pellucida genes and for dimerization with other bHLH proteins (Millar et al., 1991; Liang et al., 1997).
The data presented describe an expression pattern for the human FIGLA gene which is consistent with a role for this transcription factor in the regulation of human ZP gene expression and also other early events in oogenesis, such as the formation of primordial follicles (Soyal et al., 2000). The expression of FIGLA was detected in ovarian follicles and MII oocytes, suggesting that it may be required for the regulation of oogenesis-related genes until the oocyte acquires maturity. Expression during preimplantation development was observed less frequently (2/7 samples). Expression of FIGLA in the single blastocyst is unlikely to be representative, since PCR results for various gene transcripts using this sample have consistently been at variance with other blastocysts tested, suggesting abnormality (data not shown). In contrast to FIGLA results, ZP2 and ZP3 transcripts were detectable from primordial staged follicles up to blastocyst stage embryos. The expression of ZP gene mRNAs in primordial follicles indicates that these mRNAs may be stored and processed later in oocyte development to form the zona pellucida (Gougeon et al., 1996). Since the novel ZP2 transcript variant reported here was not observed after the 2-cell stage, it is likely that this particular transcript is exclusively maternally derived. To our knowledge, this constitutes the first report of the detection of this human ZP2 splice variant, which consequently requires further characterization.
OCT4 transcripts were not consistently observed in human ovarian follicles and mRNA expression was restricted to the cDNA derived from the mixture of early primary and primordial follicles. Down-regulation of the OCT4 transcription factor during oogenesis has been described previously (Pesce et al., 1998); however, the results presented in our study may indicate a transient role in growing ovarian follicles.
Thus far, we have been unable to detect human FIGLA transcripts in the ovary by Northern blot analysis or by RT–PCR from adult tissues, which may be a result of a low copy-number of the transcript. The age range of the ovarian tissues used in the present study was 17–60 years (seven individuals for the commercially available samples), and 22 years for the ovarian cDNA generated locally. Since follicle number declines with age, it can be anticipated that younger, or perhaps fetal, ovarian tissue may be required for detection by this method. In agreement, murine Figla expression is barely detectable by Northern blot analysis in adult ovarian tissue, and expression peaks around 2 days after birth (Soyal et al., 2000). Murine Figla gene expression is also detected by both RT–PCR and Northern blot analysis in the testis (Liang et al., 1997), but it is not required for male reproductive function since homozygous Figla null male mice are fertile (Soyal et al., 2000). These observations, taken together with the lack of detectable human FIGLA expression in cDNA derived from the testis as described in this report, may indicate that FIGLA is not essential for human male reproductive function.
Using RT–PCR from amplified cDNA pools in conjunction with cloning and sequencing (in situ and in silico), we have been able to demonstrate thus far the specificity of expression of the FIGLA gene to the human female germline. With the availability of full sequence information, further characterization of the human FIG transcription factor and its potential role in female infertility are now possible.
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Acknowledgements |
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This work was supported by N.V. Organon.
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4 To whom correspondence should be addressed. E-mail: medjhu{at}leeds.ac.uk
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Submitted on June 18, 2002; resubmitted on August 22, 2002; accepted on October 8, 2002.
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M Hinkins, J Huntriss, D Miller, and H M Picton Expression of Polycomb-group genes in human ovarian follicles, oocytes and preimplantation embryos Reproduction, December 1, 2005; 130(6): 883 - 888. [Abstract] [Full Text] [PDF]
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H. Stoop, F. Honecker, M. Cools, R. de Krijger, C. Bokemeyer, and L.H.J. Looijenga Differentiation and development of human female germ cells during prenatal gonadogenesis: an immunohistochemical study Hum. Reprod., June 1, 2005; 20(6): 1466 - 1476. [Abstract] [Full Text] [PDF]
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G. Cauffman, H. Van de Velde, I. Liebaers, and A. Van Steirteghem Oct-4 mRNA and protein expression during human preimplantation development Mol. Hum. Reprod., March 1, 2005; 11(3): 173 - 181. [Abstract] [Full Text] [PDF]
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R. A.L. Bayne, S. J. Martins da Silva, and R. A. Anderson Increased expression of the FIGLA transcription factor is associated with primordial follicle formation in the human fetal ovary Mol. Hum. Reprod., June 1, 2004; 10(6): 373 - 381. [Abstract] [Full Text] [PDF]
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