Molecular Human Reproduction, Vol. 7, No. 6, 553-558,
June 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Expression of a testis-specific member of the olfactory receptor gene family in human primordial germ cells
1 Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
Abstract
Olfactory receptors are G protein-coupled transmembrane receptors. Genes encoding olfactory receptors constitute a large gene family of ~1000 total member genes. In mammals, a subset of member genes is specifically expressed in the testis (and not in the olfactory mucosa) and olfactory receptor proteins have been identified in elongated spermatids and mature spermatozoa of dogs. It is postulated that olfactory receptors may recognize signal molecules present in the female genital tract and play a role in chemotaxis of spermatozoa towards the oocyte. In a previous study, we identified 10 cDNA sequences, corresponding to genes specifically expressed in human primordial germ cells (PGC), by differential display. Sequence analysis revealed that one of these sequences appeared to be a member of the olfactory receptor gene family. To investigate this further, we have used degenerate oligonucleotide primers corresponding to conserved amino acid sequences of olfactory receptor proteins to amplify all the olfactory receptor genes expressed in the PGC. Sequence analysis of a total of 30 cloned sequences disclosed that one member gene, which was previously isolated from a human testis cDNA library by others, was also preferentially expressed in our PGC. Our results suggest that specific members of the olfactory receptor gene family may have a function in germ cells in the migratory phase of their life cycle.
chemotaxis/developmental gene expression/gene expression/human primordial germ cells/olfactory receptors
Introduction
Olfactory receptors are G protein-coupled transmembrane receptors which recognize and discriminate a wide range of odours. Cloning of genes encoding olfactory receptors in mammals has established a basis for molecular analysis of the diverse repertoire of olfactory receptors (Buck and Axel, 1991
). It has been found that the olfactory receptor genes constitute a large family of up to 1000 member genes distributed on different chromosomes (Rouquier et al., 1998) and that each olfactory receptor protein is encoded by a different gene.
In our previous work, we created amplified cDNA preparations from primordial germ cells (PGC) isolated from the gonads of 10 week old aborted fetuses (Goto et al., 1999). The identity of the isolated PGC was confirmed by positive staining for alkaline phosphatase activity and the germ cell-specificity of prepared PGC cDNA was verified by an abundance of transcripts for a pluripotent cell marker, OCT4 (Goto et al., 1999). As in mice, expression of human OCT4 in pluripotent cells has been verified in human ES cells (Reubinoff et al., 2000), PGC (Goto et al., 1999) and unfertilized oocytes (T.Goto et al., unpublished data). Comparison of gene expression profiles of male and female PGC with each other and with that of a whole 10 week old fetus by differential display identified a number of PGC-specific genes. Among the genes identified by sequence analysis was a member of the olfactory receptor gene family expressed in both male and female PGC but not detectable in the whole fetus (Goto et al., 1999).
This finding was rather surprising and prompted us to review the literature on these genes. Interestingly, a subset of olfactory receptor genes is specifically expressed in the mammalian testis (Parmentier et al., 1992; Vanderhaeghen et al., 1997a, 1997b). Immunohistochemical analysis in dogs found that these testis-specific olfactory receptors are localized in the cell membrane of elongated spermatids and mature spermatozoa (Vanderhaeghen et al., 1993). Based on the identification of olfactory receptor molecules on the sperm cell surface and, in addition, intracellular effector molecules downstream of G proteins in spermatozoa [summarized in (Vanderhaeghen et al., 1997b) and references therein], it is postulated that male germ cell-specific olfactory receptors may recognize molecules present in the female genital tract and guide spermatozoa towards the oocyte for fertilization.
PGC originate in the extraembryonic mesoderm of the developing embryo and actively migrate through the dorsal mesentery of the hindgut to the developing gonad (Buehr, 1997). In humans, PGC arrive at the developing gonads by 7 weeks gestation (Fujimoto et al., 1977). Soon after arrival, PGC start to differentiate into oogonia (females) or prespermatogonia (males) but, unlike in mice, human germ cell differentiation in the gonad is not well synchronized and migration of germ cells is still observed within the ovary and testis up to 12 weeks gestation (Gondos and Hobel, 1971; Motta and Makabe, 1986).
Therefore, our finding might suggest that specific olfactory receptor proteins may also have a function in migration of PGC. To address the question of whether or not a particular subset of olfactory receptor genes is expressed in the PGC, we amplified olfactory receptor gene sequences expressed in the PGC cDNA using degenerate oligonucleotide primers corresponding to the conserved amino acid sequences of olfactory receptor proteins. The amplified products were cloned and a total of 30 clones were sequenced. We found that one particular olfactory receptor gene sequence was represented in 12 of the 30 clones analysed. Sequence comparison showed the sequence to be identical to an olfactory receptor gene sequence, previously isolated from a human testis cDNA library (Vanderhaeghen et al., 1997a). Expression of germ cell-specific olfactory receptor genes in PGC in their migratory phase strongly supports a hypothesis that olfactory receptors play an important role in primordial germ cell migration.
Materials and methods
Preparation of cDNA from human PGC, oocytes, embryos and embryonal carcinoma (EC) cells
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. 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 (1989). The generation of cDNA molecules used in this work from fresh human PGC was previously described in detail (Goto et al., 1999). Briefly, 200 (male) and 500 (female) PGC were isolated from the gonads of 10 week old aborted fetuses (one male and one female) within 2–3 h after the surgical termination of pregnancy. The sex of the fetus was determined by PCR amplification of X- and Y-specific amelogenin gene sequences, using genomic DNA extracted from a small piece of the fetal lung tissue. Messenger RNA (mRNA) molecules were isolated using an oligo(dT)25 nucleotide attached to magnetic beads (Dynabeads mRNA purification kit; Dynal, UK) and resuspended in 3 µl of double-distilled water. The whole 3 µl of the resuspended mRNA were used for the synthesis of first strand cDNA and subsequent PCR amplification using SMART PCR cDNA Library Construction Kit (Clontech, Palo Alto, CA, USA), according to the manufacturer's instructions.
The cDNA preparations from human oocytes and preimplantation embryos have been previously described (Holding et al., 2000). The oocytes were classified as `failed-fertilization' oocytes since formation of the male pronucleus was not observed 16–18 h following insemination in vitro. All preimplantation embryos were healthy embryos at the right developmental stage for time in culture. The procedures for oocyte and embryo cDNA preparations were essentially the same as those used for the PGC cDNA described above.
A multipotent human embryonal carcinoma (EC) cell line, GCT 27 X-1 (Pera et al., 1989; kind gift of Dr Martin Pera, Institute of Reproduction and Development, Monash Medical Centre, Victoria, Australia), was used as the source of undifferentiated pluripotent cells and their differentiated derivatives. The undifferentiated GCT 27 X-1 cells were induced to differentiate in culture for 6 days by the presence of 5 µmol/l all-trans retinoic acid (Eastman Kodak, New York, USA) in 
-minimum essential medium (MEM)/Ham's F12 medium (Gibco BRL, Mulgrave, Victoria, Australia) supplemented with 25% conditioned medium derived from the yolk sac carcinoma cell line GCT 44 (Roach et al., 1994). At the end of culture, the differentiated GCT 27 X-1 cells were removed from the culture dish by the treatment with 0.5 ml of 0.25% trypsin for 1 min. The dissociated cells were suspended in 3 ml of -MEM medium, centrifuged and resuspended in 5 ml of -MEM medium. The undifferentiated and differentiated cells were collected as batches of 1000 cells and processed to produce amplified cDNA as described above.
Control cDNA were prepared from brain, muscle and gut, from a 10 week old whole human fetus and from mouse testis of an inbred strain, FVB/N. The mRNA from the human somatic samples was purified from total RNA extracted (Chirgwin et al., 1979) using the magnetic beads and subjected to cDNA synthesis and amplification by the SMART kit, as described above. Total RNA was extracted from the mouse testis by an RNA isolation kit (Strategene, Zvidoost, The Netherlands) and cDNA was synthesized by the MMLV reverse transcriptase with random hexamer primers, according to the manufacturer's instructions (Gibco BRL, Paisley, UK).
PCR amplification of olfactory receptor gene cDNA
PCR was performed using degenerate oligonucleotide primers as described (Vanderhaeghen et al., 1997b). The sequences of these degenerate primers are: hOR-1, 5'-TGGCITA(T/C)GA(T/C) (C/A)GIT(A/T) (T/C)GTIGC-3'; and hOR-2, 5'-(A/G)AAIGG (A/G)TTNAGCATNGG-3' (where I is inosine).
The primers, hOR-1 and hOR-2, were designed to anneal with the highly conserved amino acid sequences, MAYDRYVAIC and PMLNPF, of the third and seventh transmembrane regions of olfactory receptors, respectively (see Figure 1). PCR amplification was conducted in 25 µl of reaction mixture, containing 1 µl of cDNA (concentrations were adjusted for the ß-actin PCR product) (Goto et al., 1999), 200 µM each of dNTPs, 25 pmol each of primers, hOR-1 and hOR-2, and 1.25 U Taq polymerase (Perkin-Elmer, New Jersey, USA) in the supplied buffer. The PCR cycle conditions were 35 cycles of denaturation for 1 min at 93°C, annealing for 2 min at 55°C and extension for 3 min at 72°C, followed by the final extension for 10 min at 72°C. The PCR product (10 µl aliquot) was electrophoresed on a 1.2% agarose gel in 1xTris borate EDTA buffer. The expected size of the PCR product was 525 bp.
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PCR amplification of the sequence HT2, an olfactory receptor gene isolated from a human testis cDNA library, was performed as described (Vanderhaeghen et al., 1997a
). The sequences of the HT2-specific PCR primers are: HT2-1, 5'-TGGGTGCCATATTTGGCTGT-3'; and HT2-2, 5'-AGGAGGCAGAATTTGCAGGC-3'. PCR amplification was carried out in 25 µl of reaction mixture, containing 1 µl of cDNA (concentrations were adjusted by ethidium bromide staining of diluted aliquots compared with standard DNA), 200 µmol/l each of dNTPs, 25 pmol each of primers, HT2-1 and HT2-2, 5% dimethylsulphoxide (DMSO) and 1.25 U Taq polymerase (Perkin-Elmer) in the supplied buffer. The PCR cycle conditions were an initial step of denaturation for 2.5 min at 93°C, followed by 35 cycles of denaturation for 1 min at 93°C, annealing for 2 min at 58°C and extension for 3 min at 72°C. The expected size of the PCR product was 410 bp.
Cloning of PCR products and DNA sequencing
The PCR products were excised from the gel, purified by QIAEXII kit (Qiagen, UK) and cloned into a cloning vector, pGEM-T Easy (Promega, UK). 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.
Results
In this study, we investigated whether particular members of olfactory receptor genes are preferentially expressed in the PGC. We employed the strategy of using degenerate PCR primers (Vanderhaeghen et al., 1997b) within the olfactory receptor gene to amplify expressed sequences in the PGC cDNA. The degenerate primers were designed to anneal with the cDNA sequences corresponding to conserved amino acid sequences of the third and seventh transmembrane regions of olfactory receptor proteins (Figure 1
and see Materials and methods for primer sequences). These amino acid regions are highly conserved within and across the mammalian species and have been shown to amplify a large number of olfactory receptor gene cDNA (Vanderhaeghen et al., 1997b; and see below).
We postulate that, since there are up to 1000 total member genes for olfactory receptors in each mammalian species, we would expect that sequence analysis of a limited number of PCR clones (30 in this study) will show different gene sequences at an equal frequency (probably one each of 30 different sequences), if none of them is preferentially expressed. By contrast, if some member genes are preferentially expressed, their sequence will appear at a higher frequency than others. This hypothesis is based on the assumption that the degenerate PCR strategy used can amplify the olfactory receptor genes without preference for some over others (see below).
The degenerate primer PCR produced amplification products from both male and female PGC cDNA with only a faint band from the whole fetus cDNA (Figure 2). Germ-cell-specific expression of olfactory receptor genes in the PGC is consistent with our previous identification of a germ-cell-specific olfactory receptor gene sequence by differential display of germ cell and somatic cell cDNAs (Goto et al., 1999). The faint olfactory receptor gene PCR band in the whole fetus cDNA (Figure 2, lane 3) may result from the presence of expressed sequences in PGC and nasal sensory neurons at a low level within the total cDNA preparation. Amplification of a product from a mouse testis cDNA (Figure 2, lane 4) further confirms that our degenerate PCR strategy was successful in the isolation of germ-cell-specific olfactory receptor gene sequences.
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The amplified PCR products in PGC, excised as a single band at 525 bp from the gel in Figure 2
, were cloned and a total of 30 clones (16 from male, and 14 from female, PGC) were sequenced. The results showed that one particular gene sequence was obtained frequently (12 out of 30 clones, comprising six each from male and female PGC), indicating that a particular olfactory receptor gene is preferentially expressed in PGC. Sequence comparison showed that the sequence of this PGC-specific olfactory receptor gene is identical to that of an olfactory receptor gene clone, HT2, registered on databases (accession number Y10529) (Vanderhaeghen et al., 1997a). Significantly, the HT2 clone was previously isolated from a human testis cDNA library (Vanderhaeghen et al., 1997a), using a human olfactory receptor gene probe (HGMP07) which specifically hybridizes testis-specific olfactory receptor genes in dogs (Parmentier et al., 1992). Other clones isolated from the PGC matched related olfactory receptor genes such as OR1D4 (accession number AF087922) (Glusman et al., 2000) and the OR7E14P pseudogene (accession number AF065856) (Buettner et al., 1998), none of which have been isolated from the testis. Unlike the HT2 sequence, these sequences did not appear more than once indicating that they were not preferentially expressed in PGC.
It should be noted that the identification of the clone, HT2, is not due to an artefact with the experimental strategy used. First, the degenerate PCR strategy designed by Vanderhaeghen et al. (1997b) does not normally preferentially amplify this sequence. In fact, these workers sequenced a total of 200 PCR clones amplified from human, mouse, rat and dog testis cDNA and found 77 different sequences (18 from human, 22 from mouse, 25 from rat and 12 from dog). Significantly, the HT2 clone was not present in these 200 clones analysed, confirming that the degenerate PCR strategy used in this study does not preferentially amplify this sequence. The HT2 clone was isolated by screening a human testis 
GT11 cDNA library with another human olfactory receptor sequence, HGMP07, as a probe (Parmentier et al., 1992; Vanderhaeghen et al., 1997a; see above). Second, our procedures of mRNA isolation from the samples and synthesis and amplification of cDNA are based on oligo(dT) nucleotides and not sequence-specific primers, thus ensuring no enrichment of any particular members of olfactory receptor gene family, nor any other specific gene, in the cDNA preparations obtained. Third, although olfactory receptor genes are intron-less genes, there is no concern about genomic contamination of the cDNA preparations. In a controlled experiment (in the presence and absence of reverse transcriptase) using human embryonal carcinoma (EC) cells, we have shown that our cDNA preparations are free from genomic DNA contamination (T.Goto and M.Monk, unpublished data).
To confirm the specificity of expression of the HT2 gene in PGC, we prepared HT2 sequence-specific PCR primers, which did not show cross-hybridization with other olfactory receptor genes (Vanderhaeghen et al., 1997a) (see Materials and methods for sequences). PCR amplification confirmed its expression in both male and female PGC (Figure 3). To obtain further evidence for the germ-line specificity of HT2 expression, we screened replicate cDNA prepared from human failed-fertilization oocytes and individual preimplantation embryos at the 8-cell and blastocyst stages (Holding et al., 2000), together with cDNA prepared from PGC (Goto et al., 1999), EC cells (T.Goto and M.Monk, unpublished data) and control somatic cells (from fetal brain, muscle and gut; b, m, g in Figure 3). The results showed that the HT2 gene is not expressed in the preimplantation embryos, EC cells and somatic cells, but it is expressed in the female and male PGC as expected. Somewhat surprisingly, we also found expression in the failed-fertilization oocyte samples (Figure 3, see also Discussion). The PCR products amplified from the male and female PGC and oocyte samples were excised from the gels as a single band at 410 bp, cloned and sequenced (three clones per sample). Sequence analysis confirmed that they were all identical to the HT2 sequence. These results strongly suggest the germ-line specificity of expression of the olfactory receptor gene, HT2, in PGC soon after their arrival in the gonads and thus presumably during the migratory phases of germ line development.
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Discussion
The discovery of a PGC-specific member of the olfactory receptor gene family by differential display in our previous work (Goto et al., 1999), and the identification of sperm-specific olfactory receptor genes and proteins by others (Parmentier et al., 1992; Vanderhaeghen et al., 1993), prompted us to carry out a more comprehensive search of PGC-specific olfactory receptors. In this study, we used PCR with degenerate oligonucleotide primers to amplify all the olfactory receptor genes that are expressed in our PGC samples. We found that a particular olfactory receptor gene, HT2, which was originally isolated from a human testis cDNA library (Vanderhaeghen et al., 1997a), was preferentially expressed in the PGC. The identification of expression of an olfactory receptor gene (HT2) in PGC and testis (presumably in the germ cells) by two independent research groups (ourselves and Vanderhaeghen et al., 1997a) using two different experimental procedures (degenerate PCR for PGC and library screening for testis) strongly support the hypothesis that this olfactory receptor has a special function in germ-line development. It has been postulated that some olfactory receptor molecules play a role in motility/chemotaxis of spermatozoa in the female genital tract (Vanderhaeghen et al., 1993). Our finding of the presence of transcripts of the particular olfactory receptor gene in PGC suggests a role also in the migration of PGC to the developing gonad.
Sperm chemotaxis has been widely documented in invertebrates and vertebrates (Ram et al., 1996; Eisenbach and Tur-Kaspa, 1999), but the evidence has mainly come from invertebrate species where fertilization occurs outside the body. In mammals, including humans, no specific molecules in the female genital tract (in the uterus, oviduct and follicular fluid) have been identified as an unequivocal chemoattractant of spermatozoa (Eisenbach and Tur-Kaspa, 1999). This is probably because different researchers use different experimental conditions and because the underlying mechanisms of sperm chemotaxis may be more complex than generally considered and may involve more than one ligand-receptor system (see below).
The migration of human PGC was first described by studying the serial histological sections of fetuses at different gestational ages (Witschi, 1948). Later, active migration of isolated human PGC in culture was documented (Kuwana and Fujimoto, 1983). More recently, the role of several molecules, such as transforming growth factor (TGF) ß1, integrin receptors and c-kit, in PGC migration has been investigated at the molecular level in mice but their role still remains to be elucidated (Buehr, 1997).
The failure to identify a specific chemoattractant-ligand system involved in the chemotaxis of spermatozoa and PGC suggests that the underlying mechanisms of chemotaxis are highly complex. For example, spermatozoa and PGC may recognize more than one signal at a particular location of their chemotactic pathways and/or recognize different signals at different locations. The presence of sperm- and PGC-specific olfactory receptors within the large olfactory receptor gene family makes the olfactory receptor a new candidate molecule for the recognition of chemoattractants in the chemotaxis of spermatozoa and PGC and encourages further investigation of this gene family.
Detection of HT2 transcripts in the unfertilized oocyte cDNA samples is intriguing. These cDNA were prepared from failed-fertilization oocytes defined by absence of the formation of two pronuclei. Oocytes which have not been incubated with spermatozoa are not available as they are used for treatment of infertility. Failed-fertilization oocytes were selected from a large cohort of oocytes, none of which was fertilized due to poor-quality spermatozoa, and there was no evidence of the formation of a male pronucleus. Nevertheless, it is possible that some failed-fertilization oocyte samples may have been penetrated by spermatozoa and the HT2 transcripts detected may have been derived from those carried over by spermatozoa. We have previously shown evidence for the presence of a Y-chromosome-specific sequence in failed-fertilization oocytes (Daniels et al., 1997). Alternatively, mRNA for the HT2 olfactory receptor gene present in unfertilized oocytes may have no function in the oocyte itself but is simply carried over from the PGC stage. We have previously shown evidence for persistence of mRNA for the imprinted SNRPN gene from earlier stages of oogenesis in the mature oocyte (Huntriss et al., 1998).
Further work on the characterization of the gene structure of this PGC-specific olfactory receptor gene, HT2, and functional analysis of the HT2 receptor protein by transfection assays will help to clarify these issues. The elucidation of the promoter sequence and other regulatory elements of the HT2 gene may identify PGC-specific regulatory sequences and binding proteins to such sequences. It will be interesting to investigate expression of the HT2 receptor protein in human cultured PGC-derived cells (Shamblott et al., 1998) which may lead to the establishment of an in-vitro assay system to investigate candidate chemoattractant molecules. The results are bound to lead to significant insights into the two major migratory phases, spermatozoa and PGC, in the life cycle of germ cells.
Acknowledgements
Tetsuya Goto was the recipient of a HART fellowship. The research is supported by the Birth Defects Foundation. We are grateful to Dr Martin Pera for human embryonal carcinoma cell line, GCT 27 X-1.
Notes
2 Present address: Centre for Early Human Development, Institute of Reproduction and Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia 
3 To whom correspondence should be addressed. E-mail: mmonk{at}ich.ucl.ac.uk
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Submitted on December 11, 2000; accepted on March 23, 2001.
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