Molecular Human Reproduction, Vol. 5, No. 9, 851-860,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of embryo development |
Identification of genes expressed in human primordial germ cells at the time of entry of the female germ line into meiosis
1 Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, and 2 Department of Obstetrics and Gynaecology, University College London Medical School, 86–96 Chenies Mews, London WC1E 6HX, UK
Abstract
In mammals, primordial germ cells (PGCs) are first observed in the extraembryonic mesoderm from where they migrate through the hindgut and its mesentery to the genital ridge to colonize the developing gonads. Soon after reaching the gonads, the female PGCs enter meiosis, while the male PGCs are arrested in mitosis and enter meiosis postnatally. To gain an insight into the molecular events controlling human germ cell development, we determined specific profiles of gene expression using cDNA prepared from PGCs isolated from male and female fetal gonads at 10 weeks gestation, when female PGCs start to enter meiosis. The identity of the isolated PGCs, and the cDNA molecules prepared from them, was confirmed respectively, by alkaline phosphatase staining and by the presence of transcripts of OCT4, a marker gene for PGCs and pluripotent stem cells in mice. Using differential display to compare the profiles of gene expression of male and female germ cells with each other and with that of a whole 10 week old fetus, we have identified eight transcripts differentially expressed in male and/or female germ cells. Among these transcripts, we have identified a member of the olfactory receptor gene family, which contains genes known to be germline-specific in the dog and possibly associated with chemotactic function. Another transcript is common to a previously isolated sequence from the human testis and we have extended this sequence towards the 5' end for partial characterization. The germline-specific sequences also include two novel sequences not represented in the databases. These findings are highly encouraging for the elucidation of the genetic programming of male and female germ line development.
developmental gene expression/differential display/human development/human primordial germ cells/meiosis
Introduction
Early germ cell differentiation in mammalian embryogenesis has long been a subject of great fascination (Snow and Monk, 1983
; McLaren, 1995; Buehr, 1997). Studies on the emergence and migration of primordial germ cells (PGCs) in mice, using alkaline phosphatase enzyme activity as a germ cell-specific marker (Chiquoine, 1954; Fujimoto et al., 1977; Ginsburg et al., 1990), have shown that PGCs first appear in the extraembryonic mesoderm just above the amnion at ~7 days post-coitum (d.p.c.). They are then located in the posterior end of the primitive streak at 7.5 d.p.c. at the base of the allantois and become incorporated into the hind gut at 8.5 d.p.c. (Ginsburg et al., 1990). From the hind gut, they migrate through the dorsal mesentery towards the genital ridge and begin to colonize the developing gonad at 10.5 d.p.c. (Snow and Monk, 1983; McLaren, 1995; Buehr, 1997).
In the gonad, the development of germ cells is markedly different in male and female fetuses. Mouse female PGCs enter meiosis in the fetal gonads at 12.5 d.p.c. and meiosis continues during fetal development until arrested at the diplotene stage of the first meiotic division by 5 days post-partum (dpp) (Schultz, 1986). All female germ cells remain arrested in diplotene of meiosis I until hormonal stimulation for ovulation occurs after puberty. By contrast, mouse male PGCs are arrested in mitosis in the 12.5 d.p.c. male fetus and remain in mitotic arrest until around 6 dpp, when they resume mitotic proliferation. After several rounds of mitotic division, male germ cells, now called primitive spermatogonia, become stem cells for production of type-A spermatogonia, which continue to divide until they enter meiosis of spermatogenesis (Bellvé et al., 1977).
In humans, PGCs are identified in the hind gut at 4 weeks gestation and then migrate to colonize the developing gonads by 7 weeks gestation (Witschi, 1948; Gondos et al., 1971; Fujimoto et al., 1977; Motta and Makabe, 1986). At ~10 weeks gestation, female PGCs start to enter meiosis, while male PGCs continue to divide mitotically until they are arrested in mitosis at 16–18 weeks gestation (Gondos and Hobel, 1971).
Cytological observations in mice and humans suggest that both male and female PGCs are equally capable of entering meiosis at this critical stage of fetal development (12 d.p.c. in mice and 10 weeks gestation in humans, see above) and the decision of cell fate of germ cells, either mitosis or meiosis, takes place in the developing gonads (Luciani et al., 1977; McLaren, 1995; McLaren and Southee, 1997). It is not known whether entry into meiosis at this fetal stage is induced in the female gonad, or whether it is intrinsically programmed in both male and female PGCs and inhibited in the male gonad and allowed in the female gonad, or whether it is due to a combination of these regulatory factors. Whatever the mechanisms, different genetic programming must operate in the female and male PGCs, i.e. in female PGCs entering meiosis and male PGCs entering mitotic arrest, at this critical stage. In the nematode, Caenorhabditis elegans, the cell fate decision of germ cells in the gonad, the switch from mitosis to meiosis, is controlled by key protein molecules, GLP-1 and LIN-12 (Kimble et al., 1998). In mammals, the underlying molecular mechanisms governing the different developmental pathways of male and female primordial germ cell differentiation are unknown.
In this paper, we investigate the specific profiles of gene expression in purified human fetal germ cells isolated from the gonads of a male fetus and a female fetus at 10 weeks gestation, when the female germ cells, but not the male germ cells, start to enter meiosis (Gondos and Hobel, 1971; Gondos et al., 1971, 1986). Following confirmation of their identity by alkaline phosphatase staining, we prepared cDNA from these tiny samples and demonstrated the presence of transcripts of OCT4, a transcription factor previously shown to be specifically expressed in mouse PGCs and pluripotent stem cells (Schöler et al., 1989; Palmieri et al., 1994). Using polymerase chain reaction (PCR)-based differential display (Liang and Pardee, 1992, 1997), we compared gene expression profiles of the human male and female PGCs, with reference to that of somatic cells in a human 10 week old whole fetus. We identified eight cDNA sequences which were differentially expressed in the purified PGCs, i.e. present in the germ cells but not in the somatic cells. Among the eight sequences, one was a member of the olfactory receptor gene family, some members of which are known to be specifically expressed in germ cells in the testis of the dog and mouse and may have a chemotactic function (Vanderhaeghen et al., 1997a). Another interesting germ cell-specific fragment, showing homology to a sequence previously derived from the human testis, was extended towards its 5' end by 5' RACE (rapid amplification of cDNA ends), using the original cDNA as a template sequence, and partially characterized. Other germ cell-specific sequences were entirely novel and are not represented in the databases. Further characterization of these known and novel sequences will shed new light on the genetic programming of human female meiosis and germline development, which was previously inaccessible to study.
Materials and methods
Fetal samples
The human fetal samples were obtained from the Human Embryonic Tissue Bank maintained at the Institute of Child Health (ICH) in collaboration with the Department of Obstetrics and Gynaecology, University College London (UCL), UK, and funded by the Medical Research Council. The collection, deposition and use of human fetal samples were approved by the Joint UCL/UCLH Committees on the Ethics of Human Research and the Ethical Committee of the ICH, and were carried out in accordance with the Polkinghorne report. In this study, we obtained gonads from a male and a female fetus, at 10 weeks gestation, for isolation of PGCs. The age of the fetus was the anatomical (embryonic) age, as determined by limb development, and not the age from the last menstrual period (LMP). The fetal samples were kept on ice in Leibovitz's L15 medium (Gibco BRL, UK) and the gonads were dissected from the fetuses within 2–3 h after the surgical termination of pregnancy.
The sex of the fetus was first determined by the appearance of the external genitalia and it was confirmed by PCR amplification of the amelogenin gene, which gave distinguishable X- and Y-specific amplified products. For sexing, genomic DNA was isolated as previously described (Goto et al., 1998) from a small piece of the fetal lung tissue and PCR was carried out according to a previously published protocol (Daniels et al., 1997).
Isolation of germ cells from the gonad
Germ cells were isolated from the gonad according to a previously described method (Buehr and McLaren, 1993). The gonads, dissected from the fetus, were freed from the attached mesonephric tissue under the dissecting microscope. The gonads were then incubated in 1 mmol/l EDTA in Ca2+- and Mg2+-free phosphate-buffered saline (PBS) for 5 min at room temperature to loosen the germ cells from somatic cells. The gonads were washed briefly in PBS, transferred to fresh PBS and gently squeezed by watchmaker's forceps to release the germ cells. The germ cells could be distinguished from somatic cells by their size, round shape and bright appearance. The germ cells were manually collected by a finely-drawn Pasteur pipette, and 200 (male) and 500 (female) germ cells placed in 30 µl of ice-cold lysis buffer [0.8% Igapel (Sigma, UK), 1 IU of RNase inhibitor (Gibco BRL, UK), 5 mM dithiothreitol (DTT; Gibco BRL)], snap-frozen in liquid nitrogen and stored at –70°C until RNA extraction.
A small aliquot of the isolated germ cell samples was air-dried on a glass slide for alkaline phosphatase staining (as described in Buehr and McLaren, 1993).
Preparation of polyadenylated RNA
Polyadenylated RNA [hereafter referred to as messenger RNA (mRNA)] molecules were isolated from germ cells using an oligo(dT)25 nucleotide attached to magnetic beads (Dynabeads mRNA purification kit, Dynal, UK) as follows. The germ cell samples in 30 µl of lysis buffer were mixed with 30 µl of 2x binding buffer (supplied with the beads) and incubated for 5 min at 65°C to lyse the cells and release RNA. To this, 10 µl of the oligo(dT)25-magnetic beads in suspension were added, the sample was left at room temperature for 30 min and then the beads were collected with the magnetic station (provided with the beads). The beads were washed once in 50 µl of 1x washing buffer (supplied with the beads) and three times with 50 µl of 1x reverse transcription (RT) buffer (Gibco BRL, UK). The isolated mRNA attached to the beads was resuspended in 3 µl of double-distilled (dd) H2O. Total RNA from a 10 week old human fetus was extracted (Chirgwin et al., 1979) and diluted for selection of mRNA by the magnetic beads as described above.
cDNA synthesis and PCR amplification of cDNA
The cDNA synthesis and PCR amplification of cDNA molecules were carried out by using SMARTTM PCR cDNA Library Construction Kit (Clontech, USA), according to the manufacturer's instruction. The method is shown in Figure 1. It should be noted that cDNA synthesis was performed in solid phase, i.e. the mRNA was still attached to the beads during the RT procedure.
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The whole 3 µl of the resuspended mRNA were incubated for 1 h at 42°C in 10 µl of RT reaction mixture (Clontech, USA) with a 3' oligo(dT) primer (CDS/3' primer, Table I
) and Moloney murine leukaemia virus (MMLV) reverse transcriptase (SuperScript II RNaseH– reverse transcriptase, Gibco BRL, UK). The reaction mixture also contained the SMART oligonucleotide, which annealed with the three to five cytosine residues characteristically added by the reverse transcriptase at the 3' end of cDNA molecules synthesized and serves as a further template for the extension of the cDNA by the reverse transcriptase (see Figure 1
). This extension creates a common sequence tag (complementary to the SMART oligo sequence) at the 3' ends of the cDNA molecules synthesized by RT and makes it possible to amplify these cDNA molecules in their entirety by PCR using the CDS/3' primer and the SMART primer. The tagging also makes it possible to extend a 3' cDNA sequence towards its 5' end by 5' RACE procedure (see Results).
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PCR amplification of the cDNA was carried out using 5 µl from the above RT reaction mixture, in 100 µl of PCR mixture, with initial denaturation for 1 min at 95°C, followed by 24 cycles of 15 s at 95°C and 5 min at 68°C, and final extension for 5 min at 72°C, using the CDS/3' and SMART primers (see Table I
for primer sequences).
An aliquot (5 µl) of the amplified cDNA was electrophoresed on a 1% agarose gel to check the distribution of sizes of the cDNA molecules synthesized and amplified, and 0.1 µl was used for gene-specific PCR for two housekeeping genes, ß-actin and hypoxanthine phosphoribosyl transferase (HPRT) and a gene, OCT4, shown to be specific for pluripotent cells in mice, to verify successful RT and subsequent PCR amplification of cDNA molecules generated, and germ cell specificity. Published sequences of the PCR primers used for ß-actin, HPRT and OCT4 genes are shown in Table I. The PCR cycling parameters are 35 cycles of 1 min at 95°C, 1 min at 62°C and 1 min at 72°C for ß-actin (Heikinheimo et al., 1995) and 35 cycles of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C for HPRT (modified from Daniels et al., 1997) and OCT4 (modified from Abdel-Rahman et al., 1995).
Differential display
Differential display was performed as previously described (Liang and Pardee, 1997). The volume of PCR mixture was 20 µl, comprising 1 µl of the cDNA samples (concentrations adjusted for comparison), 2 µmol/l each of dNTPs, 0.2 µl of [
-33P]-dATP (10 mCi/ml, Amersham, UK), 0.2 pmol each of primers and 1.25 IU of Taq polymerase (Perkin Elmer, UK) in the supplied PCR buffer. Sequences of primers are shown in Table I. The random primers (H-AP1 and H-AP2, Table I) defined the size of fragment for each particular gene sequence, depending on the position [from the poly(A) tail] of homology to the random primer sequence. Primers, TG, TA and TC, were oligo(dT) primers for annealing with the poly(A) tail. One nucleotide, G, A or C, was attached to the 3' end of these oligo(dT) primers to ensure that the primer annealed at the beginning of poly(A) tail sequence. Therefore, PCR amplification with a primer set of one of the three 3' oligo(dT) primers (TG, TA or TC) and either H-AP1 or H-AP2 random primers should produce a 3' end cDNA fragment of specific length for each gene cDNA.
The PCR cycle parameters were 40 cycles of 30 s at 94°C, 2 min at 40°C and 30 s at 72°C, followed by 10 min extension at 72°C. The annealing at 40°C allowed three to four nucleotide mismatches between the random primers and cDNA (T.Goto, unpublished data) and enabled the amplification of a larger number of cDNA molecules with the limited number of primer sets.
The PCR products (2 µl) were electrophoresed on a 6% denaturing polyacrylamide gel in 1x Tris borate EDTA at 1700 V until the xylene cyanol FF dye reached the bottom of the gel. The gel was transferred onto a 3MM paper (Whattman, UK), dried and exposed to a Hyperfilm (Amersham, UK) overnight at room temperature.
Re-amplification of differentially expressed cDNA fragments
The autoradiogram was superimposed on the dried gel to locate the cDNA fragments of interest. These were excised with a sterile blade, the DNA eluted from the gel by boiling in 100 µl of dd H2O for 15 min and precipitated with 100% ethanol in the presence of 50 µg of glycogen as a carrier. The DNA was washed with ice-cold 85% ethanol and resuspended in 10 µl of ddH2O.
To obtain sufficient amount of the fragments for ligation to the cloning vector, pGEM-T Easy (Promega, UK), 4 µl of the resuspended DNA were re-amplified with the same set of primers as used for differential display and with the same PCR conditions except that the concentration of dNTPs was 20 µmol/l each, instead of 2 µmol/l, and [-33P]-dATP was omitted.
DNA sequencing
Re-amplified cDNA fragments were gel-purified by QIAEXII kit (Qiagen, UK) and cloned into the pGEM-T Easy vector. DNA sequencing was carried out using the Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham, UK) with M13 forward primer, according to the manufacturer's instructions. All fragments were sequenced on both strands.
Results
In humans, it has been demonstrated cytologically that female fetal germ cells start to enter meiosis at ~10 weeks gestation (Baker, 1963; Kurilo, 1981; Speed, 1985; Gondos et al., 1986). By contrast, male germ cells differentiate into gonocytes and are arrested in mitosis (Gondos and Hobel, 1971). Therefore, a comparison of the profiles of gene expression in male and female fetal germ cells isolated from the gonad at 10 weeks gestation will not only identify genes commonly expressed in both male and female germ cells but also those differentially controlling male and female germ cell development (Figure 1).
Isolation of fetal germ cells
Germ cells, released from the gonad, were distinguished from non-germ cells by their characteristic appearance, i.e. larger, round and bright, and were manually collected. Staining for alkaline phosphatase, which is a marker for germ cells, showed that >80% of the cells isolated were positive (Figure 2). This ensures that a comparison of gene expression profiles in this cell population with that of the total cells from a 10 week old fetus (where germ cells are in a minority compared with total somatic cells) will enable us to readily identify genes differentially expressed in germ cells at this specific time of fetal development.
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cDNA synthesis and PCR amplification of cDNA
To minimize the loss of mRNA and to produce a sufficient amount of cDNA analysed from a small number of sample cells, we selected mRNA using oligo(dT)25-attached magnetic beads and PCR-amplified the cDNA molecules synthesized by RT.
Ethidium bromide staining following electrophoresis of the amplified cDNA molecules on an agarose gel shows that the sizes of the majority of cDNA molecules synthesized and amplified by PCR range from 0.5 to 2 kb (Figure 3a). Gene-specific PCR amplification of two housekeeping genes, ß-actin and HPRT, from nucleotide positions 441–910 for the 1.8 kb ß-actin cDNA and 222–808 for the 1.4 kb HPRT cDNA, confirmed that average size (1.5–2 kb), and possibly full-length, mRNA molecules were indeed detectable in the cDNA created (Figure 3b).
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To confirm the identity of germ cell-specific cDNA molecules in the cDNA preparations, we showed the presence of cDNA molecules of the OCT4 gene in the amplified cDNA populations from both the male and female germ cells (Figure 3b
). In mice, Oct4 is known to be specifically expressed in totipotent PGCs and the inner cell mass (ICM) cells (Schöler et al., 1989; Palmieri et al., 1994). The OCT4 transcripts were also detectable in the 10 week old total fetal cDNA (which includes a small fraction of germ cell cDNA) although at a much lower level (data not shown). The primers used for these gene-specific PCRs were designed to amplify an intron-spanning region thus confirming that the amplification originates from mRNA, not from genomic DNA. The absence of amplified product corresponding to the intron-spanning genomic sequence (which should appear as larger band) also confirmed that genomic DNA contamination in the cDNA samples is negligible. For differential display described below, concentrations of cDNA samples were adjusted from the relative intensities of the ß-actin PCR product.
Screening of germ cell-specific transcripts by differential display
To identify genes differentially (quantitatively and qualitatively) expressed in either or both of the male and female germ cell samples, we employed differential display (Liang and Pardee, 1992, 1997). The differential display technique is based on PCR amplification of specific 3' sequences of the cDNA molecules from different cell populations with defined sets of PCR primers, followed by separation of the PCR products by size on a sequencing gel. Comparison of gel patterns between the samples will disclose differentially-expressed genes. Germ cell-specific transcripts will be present only in the male and/or female germ cell lanes but absent (or markedly under-represented) in the total fetal lane. If a band is present in all three lanes, it is likely that the transcript is ubiquitous, e.g. housekeeping gene transcripts.
Figure 4 shows the results of differential display with the six different sets of primers chosen. Repeat experiments of the whole differential display procedure (i.e. PCR amplification of the three cDNA samples and separation of amplified fragments by electrophoresis) gave reproducible banding patterns. Twelve of the reproducibly amplified fragments (correspondingly numbered in Figure 4 and Table II), showing differences between the samples, were excised from the gel, re-amplified with the same set of PCR primers, cloned and sequenced (Table II).
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Several clones were sequenced for each individual fragment analysed and the DNA sequences were identical for each fragment. In addition, when fragments were present in more than one lane (e.g. fragment 1 which is present in all three lanes), they were individually sequenced and, in all cases, DNA sequences were identical in the different samples. These results confirmed that each band on differential display represented a single cDNA molecule and comparison between the samples for screening of differentially-expressed genes is valid.
Nine out of the 12 fragments showed matches or high homology (>95%) with known cDNA sequences in the database (Table II). Seven of the nine (except for fragments 3 and 11) corresponded to the 3' end of the homologous mRNA, which indicated the successful selection and amplification of mRNA-derived sequences. Note the high fidelity of the procedures, as shown by the degree of sequence homology.
Characterization of 12 of the differentially-expressed genes
The sequences of fragments 1 and 2, which were present in all three lanes (male germ cells, female germ cells and total fetus), match respectively to the 3' ends of the human nascent polypeptide-associated complex subunit gene (NAC) cDNA and a gene highly homologous with the human tumour necrosis factor (TNF) receptor 1 gene. Both genes are known to be expressed in a wide range of cells.
Fragments 3, 4 and 5 were expressed in both male and female germ cells, but not in the total fetal cells. Sequencing of fragment 3 showed a 95% homology with a member of the human olfactory receptor gene family. It is known that a subset of olfactory receptors is specifically expressed in male germ cells in the testis of the dog, rat and mouse (Vanderhaeghen et al., 1997a,b). However, fragment 3 showed the highest homology to an internal sequence of a pseudogene of the olfactory receptor gene family.
Sequence analysis of fragments 4 and 5 disclosed that these two fragments were identical except that fragment 5 had an 11 bp deletion at its 3' end. A database search showed that fragment 4 matches to the sequence of two short 3' expressed sequence tag (EST) clones, accession numbers aa460929 and aa993606 (Table II). Clone aa460929 was isolated from a 9 week old total human fetal cDNA library and clone aa993606 from a human testis cDNA library. Both clones represented short 3' sequence and the full-length sequence was not registered in the database. Consequently, fragment 4 was further investigated for its full-length cDNA (see below).
The identification of ubiquitous transcripts, NAC and TNF receptor (fragments 1 and 2), in all three samples and potential germ cell-specific transcripts, olfactory receptor gene (fragment 3) and a testis-derived sequence (fragments 4 and 5) in the two different germ cell samples indicated that the procedure, from cell isolation to differential display, was reproducible and that the cDNA molecules in the samples were representative of the cells from which they were isolated.
Two female germ cell-specific fragments (fragments 6 and 7) and three male germ cell-specific fragments (fragments 8, 9 and 10) were sequenced. Two of these (fragments 6 and 8) were novel sequences, not registered in the database. One of the two female germ cell-specific transcripts was matched to a 3' end sequence containing an L1 repetitive sequence and two of the three male germ cell-specific transcripts showed homology, one with the human calreticulin cDNA and the other with the human RalBP1-associated EH domain protein 1 (Reps1) cDNA.
We also sequenced two other differentially expressed fragments (fragments 11 and 12, Table II), neither of which were germ cell-specific; fragment 11 was expressed in the male germ cells and total fetus, but markedly under-expressed in the female germ cells, and fragment 12 was present in the female germ cells and total fetus, but not in the male germ cells. Fragment 11 was matched to an uncharacterized human cDNA clone, aa843148, but fragment 12 did not match any known sequence. The expression patterns of fragments 11 and 12 were reproducible in repeated differential display procedures and may suggest differences in timing of expression of these genes in male and female germ cell development.
Extension towards the 5' end of the PGC cDNA sequence homologous to a testis cDNA
In this study, the primers used in PCR amplification for differential display were designed to amplify the 3' end of the cDNA molecules but it was possible to extend the cDNA sequences towards their 5' ends by 5' RACE using the SMART primer and the original cDNAs from the germ cell samples.
Among the 12 fragments sequenced (Table II), fragment 4 is of interest because it is germ cell-specific and a 375 bp 3' EST with the identical sequence has previously been isolated from a human testis cDNA library (see Table II). To obtain the 5' region of the cDNA corresponding to the 3' differential display sequence of fragment 4, we conducted 5' RACE on the cDNA samples using the SMART primer with an internal primer, pB1, from within the sequence of fragment 4 (see Figure 5 for location). As expected from the results of the differential display, this produced a discrete PCR fragment from both male and female germ cell samples, but not from the total fetal sample (data not shown). The identity of this PCR fragment was confirmed by Southern blotting using an oligonucleotide, pB2, immediately upstream to pB1 within fragment 4 (Figure 5), as a probe (data not shown). The 5' PCR fragment, corresponding to the 3' fragment 4, was cloned and several clones were sequenced. Sequence analysis showed that all these clones were identical and that the sequence corresponding to the previously isolated EST clones was also identical. The total length of the constructed putative full-length cDNA is 641 bp and the longest open reading frame (ORF) consisted of 114 amino acids (Figure 5). The first methionine was within a sequence favourable for the initiation of translation (Kozak, 1991). This protein did not contain any known characteristic motif but there was one putative phosphorylation site for protein kinase C (double underlined in Figure 5).
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Discussion
Early human fetal germ cell development has been investigated cytologically (Gondos and Hobel, 1971; Gondos et al., 1971, 1986; Fujimoto et al., 1977; Motta and Makabe, 1986) but, to our knowledge, there are no reports on gene expression in human PGCs. Therefore, our study is the first attempt to investigate the profile of differential gene expression in human germ cell samples isolated from male and female fetal gonads. Purity of the germ cell samples was confirmed by positive alkaline phosphatase staining of the cells and the cDNA preparations were shown to be germ cell-specific by the abundance of cDNA molecules representing transcripts of the transcription factor gene, OCT4. In mice, Oct4 is expressed only in PGCs (both male and female), unfertilized oocytes and ICM of the blastocyst (Schöler et al., 1989; Palmieri et al., 1994). Our result shows that OCT4 is expressed in human PGCs, as in mouse PGCs.
The earliest cytological differences in differentiation of PGCs between males and females are observed at ~10 weeks gestation (Gondos and Hobel, 1971; Gondos et al., 1971, 1986), when the female germ cells, but not the male germ cells, start to enter meiosis (Baker, 1963; Kurilo, 1981; Speed, 1985). Differentially expressed transcripts were identified by differential display, comparing gene expression profiles between male and female germ cells and somatic (total fetal) cDNA samples. We identified two germ cell-specific (both male and female), two female germ cell-specific and three male germ cell-specific transcripts.
Sequencing analyses of two germ cell-specific transcripts (present in both male and female germ cells, but not in a total fetus) showed that one is a member of the human olfactory receptor gene family. Identification of expression of an olfactory receptor gene is interesting. The olfactory receptor is a G-protein coupled transmembrane receptor with no known specific ligand. The olfactory receptor genes are evolutionarily conserved with a gene family of up to 1000 member genes with 70% of them as pseudogenes (Rouquier et al., 1998). Importantly, a particular subset of the olfactory receptor gene family is known to be specifically expressed in the testis of dogs, rats and mice, and transcripts of some of the olfactory receptor gene family are also detected in the human testis (Vanderhaeghen et al., 1997a,b). In the dog, the protein products of this gene subset are located by immunohistochemistry to round and elongated spermatids and even mature spermatozoa. It is postulated that these protein molecules may be involved in the chemotactic movement of spermatozoa in the female genital tract (Vanderhaeghen et al., 1993). The transcript identified in this study is most likely to be one of the pseudogenes, some of which are known to be transcribed but prematurely truncated (Rouquier et al., 1998). Our finding of the expression of a subset of olfactory receptor genes in primordial germ cell development might suggest their involvement in migration. Since the transcript identified in this study was isolated by chance with only a few sets of PCR primers, a more thorough screening of the germ cell cDNA samples is likely to identify more functionally expressed germ cell-specific olfactory receptor genes.
Another germ cell-specific transcript identified by differential display is a novel gene of unknown function, previously isolated as a short 3' sequence in a human testis cDNA library. We were able to elongate the sequence towards a full-length cDNA to produce a 641 bp cDNA with a longest ORF of 114 amino acids. Analysis of the amino acid sequence reveals that this protein has no homology to known proteins in the databases and the only motif identified is a phosphorylation site for protein kinase C (PKC). It is likely that this protein is a novel effector in the PKC-dependent signalling pathway involved in the maintenance of the germline in both males and females.
Two female germ cell-specific transcripts do not match any known gene sequences although one shows homology with the 3' end of an L1-containing sequence. With regard to male germ cell-specific transcripts, two out of three are identical to portions of known human cDNA sequences, namely, the calreticulin and Reps1 genes. Calreticulin, a major Ca2+-binding protein in the lumen of the endoplastic reticulum, regulates the intracellular Ca2+ concentration (Fliegel et al., 1989; Milner et al., 1991; Camacho et al., 1995). Although calreticulin is expressed in a wide range of cells (Milner et al., 1991), the level of transcription of the calreticulin gene in germ cells has not been specifically investigated. Significantly, another Ca2+-binding protein present in the membrane of the endoplasmic reticulum, calmegin, is male germ cell-specific and required for production of spermatozoa capable of binding to the zona pellucida of eggs in mice (Ikawa et al., 1997). These results may suggest that the regulation of intracellular Ca2+ concentration plays a role throughout male germ cell development and that calreticulin has a male germ cell-specific function in the control of cell cycle at this stage of fetal development. The other male germ cell-specific transcript encodes a recently identified protein, Reps1 (RalBP1 associated Eps domain-containing protein) (Yamaguchi et al., 1997). The function of Reps1 is not yet clear but it is considered that this protein is involved in an intracellular signal transduction pathway triggered by epidermal growth factor (EGF) (Yamaguchi et al., 1997). It has been reported that several other growth factors, e.g. stem cell factor (SCF), leukaemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF), are required for the survival and proliferation of murine PGCs in culture (De Falici and Dolci, 1991; Dolci et al., 1991; Godin et al., 1991; Matsui et al., 1992; Resnick et al., 1992). In the male fetal gonad, EGF, in association with Reps1, may have a special function in maintaining the male germ cells in mitosis.
Our study has great implications for future molecular analyses of human fetal germ cells. Human fetal germ cell cDNA, and/or cDNA libraries constructed from them, will be a valuable resource for those researchers interested in this fascinating field of research. They offer the possibility of elucidating the patterns of gene expression in the regulation of meiosis, and responses of germ cells to inducer and/or inhibitor of meiosis (McLaren, 1995) and identifying marker molecules specific for early germ cells. They may also be used for more specific investigations, such as the timing and mechanisms of erasure of genomic imprinting and the mechanism(s) of reactivation of the inactive X chromosome (Gartler and Goldman, 1994; Goto and Monk, 1998).
Acknowledgments
This paper is dedicated to late Professor Peter Thorogood at the Department of Developmental Biology, Institute of Child Health, London, with gratitude for his encouragement and support for this work. We are grateful to the Human Embryonic Tissue Bank for providing us with human fetal gonads. We thank Ms Rachel Moore for the help with dissecting the gonads from the fetus. Tetsuya Goto is the recipient of HART fellowship. Marilyn Monk and James Adjaye are supported by the Medical Research Council.
Notes
3 To whom correspondence should be addressed at: Molecular Embryology Unit, Institute of Child Health,30 Guilford Street, London WC1N 1EH, UK 
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Submitted on March 17, 1999; accepted on June 1, 1999.
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