Four zona pellucida glycoproteins are expressed in the human.

BACKGROUND: The zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the study was to determine if all four ZP proteins are expressed and present in the human. METHODS: cDNA derived from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). RESULTS: All four genes were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed that the mouse ZPB gene, although present, is likely to encode a non-functional protein. CONCLUSIONS: Four ZP genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corresponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse model

Gene expression studies on human primordial germ cells and preimplantation embryos.

Research on the regulation of gene expression in human germ cells and preimplantation embryos is restricted due to the scarcity of samples and the requirement for highly sensitive molecular techniques to investigate the few cells available. To overcome these difficulties, we have developed a reliable procedure capable of generating amplified cDNA preparations from single cells. Using this procedure, we prepared cDNA from primordial germ cells (PGCs) isolated from the gonads of fetuses at 10 weeks gestation and from preimplantation embryos at the 1-cell, 4-cell, 8-cell and blastocyst stages. Our cDNA preparations allow us to investigate the expression profile of an almost unlimited number of different genes in the same sample preparation. This is of great advantage for studies of a panel of genes in a particular family or functional group, or with related mechanisms of regulation, e.g., developmental genes, oncogenes, cell cycle-control genes and imprinted genes. We have used these cDNA preparations in conjunction with differential display to identify genes specifically expressed in PGCs and preimplantation embryos in a sex- and developmental stage-specific manner. Genes specifically expressed in PGCs, oocytes and embryos were further analysed for their expression in embryonal carcinoma cells and in their differentiated derivatives following treatment by retinoic acid. Our strategy will disclose genes essential for gametogenesis and embryonic development which may only be expressed at certain stages of their development. The germ cell- and embryo-specific cDNA molecules, cDNA libraries and microarrays are a valuable resource for other researchers in this field.

Expression of imprinted genes in human preimplantation development.

Imprinted gene expression in preimplantation development has been extensively studied in the mouse. Different imprinted genes vary in their time of onset of expression and also in the timing and tissue-specificity of mono-allelic expression. We have surveyed a range of imprinted genes for expression, and mono-allelic expression, in human development. Due to the scarcity of human embryos available for research, we first prepared amplified cDNA from replicate samples of human oocytes, four-cell, eight-cell and blastocyst stages. We then analysed these cDNAs for expression of a range of imprinted genes. Three of six genes analysed (SNRPN, PEG1 and UBE3A) are clearly expressed in preimplantation embryos. Expression was confirmed by direct analysis of embryos for these genes. For one of the expressed genes, SNRPN, we have shown that expression is mono-allelic from the paternal allele in human preimplantation embryos. This gene is also mono-allelically expressed in mouse preimplantation embryos. In our earlier work, we investigated the molecular mechanisms governing mono-allelic expression of the paternal allele of the Xist gene in preimplantation mouse embryos. We found that mono-allelic expression was correlated with differential methylation of Xist promoter sites in egg and sperm, and specific binding of a protein only to the methylated maternal (egg) allele. However, extension of these studies to the human showed that, unlike the mouse, XIST is expressed from both parental alleles in human preimplantation embryos. Since perturbation of imprinting is associated with disease and tumourigenesis, it is important to know the expression profiles of imprinted genes in human embryos and to monitor for normal imprinted gene expression with the introduction of new procedures in assisted conception.

The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos.

Imprinted genes are characterized by expression of only one of the two alleles according to its inheritance from the mother or the father. This mono-allelic expression must arise from primary differential epigenetic modification of the parental alleles of the imprinted gene in the spermatozoon and the oocyte. Most of the information on the onset of imprinted gene expression, and on the molecular mechanisms regulating mono-allelic expression, have been derived from studies in the mouse. In this paper, we investigate the expression of seven imprinted genes in human preimplantation development. Due to limitations imposed by the rarity of human embryos available for research, our approach has been to screen amplified cDNA preparations prepared from human unfertilized oocytes and individual embryos at each of the 4-cell, 8-cell and blastocyst stages. Gene-specific primers were used to investigate expression of the imprinted genes by polymerase chain reaction (PCR) analysis of these amplified cDNA. We found that expression is inherently variable in the amplified cDNA from embryo to embryo but the use of several samples at each stage showed that the SNRPN, UBE3A and PEG1 genes are expressed throughout human preimplantation development. This was confirmed by direct analysis by gene-specific reverse transcription-PCR on a limited number of lysed embryos (one gene analysed per embryo). Thus, the amplified cDNA may be used to rapidly identify those imprinted genes expressed in preimplantation development and, hence, those genes amenable to investigation of the epigenetic mechanisms regulating mono-allelic expression. Confirmation of preimplantation expression also identifies those imprinted diseases amenable to preimplantation diagnosis, and the imprinted genes which may be used in assessment of possible perturbations of imprinting following new procedures in assisted reproduction. Our series of single embryo amplified cDNA are established as a valuable resource for comparative studies of gene expression within one embryo and between embryos throughout early human development. The amplified cDNA thus circumvent the need for a continuous supply of human embryos for studies on embryonic gene expression.

Expression of a testis-specific member of the olfactory receptor gene family in human primordial germ cells.

Olfactory receptors are G protein-coupled transmembrane receptors. Genes encoding olfactory receptors constitute a large gene family of approximately 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.

Identification of differential methylation of the WT1 antisense regulatory region and relaxation of imprinting in Wilms' tumor.

Wilms' tumor (WT) is associated with loss of heterozygosity at chromosome 11p13, the site of the Wilms' tumor suppressor gene, WT1. Although the preferential loss of maternal alleles suggested that differential allelic expression of WT1 might occur, this has not been evident in normal fetal tissues or WTs. In this study, we show that the WT1 antisense regulatory region is differentially methylated, with Southern blot analysis of four loss of heterozygosity-negative WTs and their corresponding normal kidneys indicating that allelic methylation is lost in WTs. Reverse transcription-PCR expression analysis correlates methylation with monoallelic expression of the antisense WT1 transcript (WT1-AS) in normal kidney. However, WTs display hypomethylation and biallelic expression of WT1-AS. Our findings are consistent with imprinting of WT1-AS in normal kidney and the relaxation of imprinting in Wilms' tumorigenesis. This identifies the WT1 antisense regulatory region in intron 1 as a primary site for epigenetic deregulation at chromosome 11p13 in WTs.

Transactivation of the WT1 antisense promoter is unique to the WT1[+/-] isoform.

The Wilms' tumour suppressor gene, WT1, encodes a zinc finger transcription factor that has been shown to repress a variety of cellular promoters via binding to cognate DNA elements. Our earlier work identified an antisense WT1 promoter that contains WT1 consensus sites, but is transcriptionally activated by WT1. In this study, we demonstrate that, unlike previous reports of transcriptional regulation by WT1, transactivation of the antisense promoter is unique to a single isoform of WT1. Of the four alternatively spliced isoforms in which exon 5 (at splice I) or amino acid residues KTS (at splice II) are inserted or omitted, only the WT1 isoform containing splice I and omitting splice II (WT1[+/-]) displays transactivation. We demonstrate that transregulation variations observed with WT1 isoforms are not solely attributable to differential DNA binding by [+KTS] or [-KTS] isoforms. Thus, the transactivation of the antisense promoter displays an absolute requirement for exon 5, suggesting that interaction between WT1 and other cellular factors is necessary for this regulatory function.

Antisense WT1 transcription parallels sense mRNA and protein expression in fetal kidney and can elevate protein levels in vitro.

Recent studies have identified antisense WT1 mRNAs whose expression is regulated by a promoter located in the first intron of the WT1 gene. Transcription directed by the antisense promoter is positively autoregulated by the WT1 protein implicating the antisense RNA in the control of WT1 gene expression. To elucidate further the biological role of the antisense RNA in the developing kidney, its distribution of expression has been examined relative to WT1 sense mRNA and WT1 protein. Using strand-specific WT1 riboprobes, the expression of WT1 and the antisense message were examined by in situ hybridization in the developing human fetal kidney at different gestational ages. The expression of the antisense strand was strongest in the podocytes and glomeruli and also in the S-form nephrons and the condensing blastema in the developing kidney. Expression was also seen in the podocytes of the mature kidney. The WT1 protein and sense mRNA for WT1 also showed a similar pattern, suggesting that the antisense transcript does not function simply as a downregulator of protein production. Expression of antisense WT1 exon 1 in cells constitutively producing high levels of WT1 also demonstrated no downregulation of protein and in most cases actually showed upregulated WT1 protein expression. These results strongly suggest that WT1 antisense transcripts positively modulate WT1 protein levels in vivo.