Cryopreservation of placental biopsies for mitochondrial respiratory analysis.

Mitochondrial function is required to support energetically-demanding processes in the placenta. As such, a compromise in mitochondrial function could severely impact fetal growth and development. Respirometry is a highly useful method for studying mitochondrial function, but is not possible in freeze-thawed mitochondria, which become uncoupled. We have developed a novel method that permits respiratory analysis of cryopreserved placental tissue. We studied mitochondrial function in 7 normal human placentas, analysing both fresh and cryopreserved samples. We found no impairments in respiration following cryopreservation in the delivery suite, with enhanced coupling, as indicated by higher respiratory control ratios, than in fresh placental samples transported to the laboratory on ice.

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

Mammalian embryonic development--insights from studies on the X chromosome.

This paper reviews our early studies on the cycle of changes of X chromosome activity in different lineages of the developing female mouse embryo. The emphasis is placed on the insights gained into key developmental processes such as the temporal and spatial aspects of developmental totipotency, the timing and cell numbers involved in the origin of the germ line, the molecular mechanisms of genetic deprogramming, reprogramming and X chromosome imprinting, and transgenerational epigenetic inheritance. When viewed in this way, it is quite remarkable to see how much was learned about mammalian development from early studies on the X chromosome. Indeed several paradigm shifts occurred as a result of these studies and these are highlighted in this review.

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.

Isolation of novel developmental genes from human germ cell, oocyte and embryo cDNA by differential display.

Due to the difficulties inherent in research on human embryos, almost nothing is known about genes active in human early development. Although the human genome project will provide resources that theoretically provide access to every human gene, those genes specific to human early development may be difficult to define. Also, by definition, genes specific to early development will not be represented in cDNA databases derived from human somatic cells. Yet these unknown human developmental genes are likely to be of key importance for several areas of human health, including assisted reproduction and contraception, embryo stem cell research and tissue transplantation, ageing and cancer. In order to identify and isolate these human developmental genes, we have prepared amplified cDNA from human primordial germ cells, oocytes and embryos, and used differential display to compare patterns of gene expression in these embryonic cells and in the cells of somatic tissues of a 10-week human fetus. This paper reviews the highly sensitive procedures used to create amplified cDNA representing expressed genes in a single cell and the use of differential display to identify developmental genes. Several such genes have been isolated, but their full-length sequences and function are yet to be elucidated. Genes active in human early development are expected to play key roles in the maintenance of the archetypal stem cell state, potential immortality and the invasiveness of trophectoderm and primordial germ cells. They represent candidate genes regulating these functions for targeting in clinical research in human reproduction, stem cell differentiation and cancer.

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.

Of microbes, mice and man.

This chapter reviews my 18 years of research in Anne's Unit including studies on temporal and spatial aspects of X-chromosome inactivation and imprinting and the role of methylation in X-inactivation in these processes during female mouse embryo development. To enable molecular studies of embryos, we developed a plethora of single cell assays for specific enzyme activity, gene mutation and methylation, and RNA transcription. While in Anne's Unit, I used these same single cell assays to pioneer the procedures for preimplantation diagnosis of genetic disease, now an established clinical approach to prevention of the birth of children with severe genetic disease. At the Institute of Child Health in London, we continue to develop new highly sensitive molecular procedures--currently for the creation of cDNA libraries from human preimplantation embryos, primordial germ cells and embryonal stem cells. We are using these cDNA preparations to isolate human developmental genes and embryo/cancer genes. One of the more fascinating aspects arising from my time in Anne's Unit is the way in which my research findings challenged a number of accepted dogmas in development concerned with the origin and totipotency of the germ line and the possibility of transgenerational genetic inheritance by epigenetic modification of the germ line.

Human embryonic genes re-expressed in cancer cells.

Human preimplantation embryonic cells are similar in phenotype to cancer cells. Both types of cell undergo deprogramming to a proliferative stem cell state and become potentially immortal and invasive. To investigate the hypothesis that embryonic genes are re-expressed in cancer cells, we prepare amplified cDNA from human individual preimplantation embryos and isolate embryo-specific sequences. We show that three novel embryonic genes, and also the known gene, OCT4, are expressed in human tumours but not expressed in normal somatic tissues. Genes specific to this unique phase of the human life cycle and not expressed in somatic cells may have greater potential for targeting in cancer treatment.

Detection of human novel developmental genes in cDNA derived from replicate individual preimplantation embryos.

We have constructed amplified cDNA preparations from replicate samples of human oocytes and individual preimplantation embryos. Differential display of the cDNA preparations shows disparate patterns of gene expression in the individual embryos at all stages of preimplantation development. The variation in patterns of genes expressed is in part due to the low starting cell number undergoing the reverse transcription-polymerase chain reaction (RT-PCR) step in the preparation of the amplified cDNAs. Despite this variability, the use of replicate embryo samples makes it possible to identify and isolate human genes specifically expressed at the different stages of human preimplantation development from the unfertilized oocyte to the blastocyst stage.

Transcription of homeobox-containing genes detected in cDNA libraries derived from human unfertilized oocytes and preimplantation embryos.

Genes containing the evolutionarily conserved homeodomain sequence encode a family of DNA-binding transcription factors whose functions are crucial for embryonic development in vertebrates, invertebrates and plants. We describe the detection and analysis of transcripts of homeobox-containing genes present in cDNA libraries generated from human unfertilized oocytes, single cleavage stage embryos (2-cell, 4-cell, 8-cell and blastocyst) and a 10-week old whole fetus. Using degenerate primers derived from sequences within helix 1 and helix 3 of the highly conserved region of the ANTENNAPEDIA:-class homeodomain, a 166 bp band was detected in all the cDNA libraries tested. Subcloning of the oocyte-derived band revealed that it contained a heterogeneous group of 166 bp fragments. Sequence analysis of 40 independent clones demonstrated the presence of HOXA7, HOXD8, and HOXD1 sequences, the ubiquitously expressed POU family member, OCT1, and HEX, a homeotic gene expressed in haematopoietic cells.

Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos.

We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.

Identification of genes expressed in human primordial germ cells at the time of entry of the female germ line into meiosis.

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.

Imprinted expression of SNRPN in human preimplantation embryos.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurogenetic disorders arising from a loss of expression of imprinted genes within the human chromosome region 15q11-q13. Recent evidence suggests that the SNRPN gene, which is defective in PWS, plays a central role in the imprinting-center regulation of the PWS/AS region. To increase our understanding of the regulation of expression of this imprinted gene, we have developed single-cell-sensitive procedures for the analysis of expression of the SNRPN gene during early human development. Transcripts of SNRPN were detected in human oocytes and at all stages of preimplantation development analyzed. Using embryos heterozygous for a polymorphism within the SNRPN gene, we showed that monoallelic expression from the paternal allele occurs by the 4-cell stage. Thus, the imprinting epigenetic information inherited in the gametes is recognized already in the preimplantation embryo. The demonstration of monoallelic expression in embryos means that efficient preimplantation diagnosis of PWS may be made by analysis for the presence or absence of SNRPN mRNA.

Detection of a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase gene in human oocytes and preimplantation embryos: implications for a RT-PCR-based preimplantation diagnosis of Lesch-Nyhan syndrome.

We have detected a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in two human oocytes and four preimplantation embryos from the 4-cell to the 8-cell stage of development. The novel HPRT transcript lacks exons 4, 5 and 6 of the normal HPRT gene. The same parental origin for the two oocytes and two of the preimplantation embryos, in which the alternatively spliced transcript was detected, might suggest that the alternative splicing is influenced by genetic background. Mutations in the HPRT gene which cause alternative mRNA splicing are implicated in Lesch-Nyhan syndrome. However, the relatively high frequency of detection of this novel HPRT transcript described here (6/109 oocytes and preimplantation embryos) suggests that it is not involved in Lesch-Nyhan syndrome. It is probable that the alternative HPRT transcript is derived from the aberrant splicing of a small percentage of the total mRNA produced from normal HPRT alleles. The presence of this alternative transcript in human preimplantation embryos may complicate an reverse transcription-polymerase chain reaction-based preimplantation diagnosis of Lesch-Nyhan syndrome.

Regulation of X-chromosome inactivation in development in mice and humans.

Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females. X-chromosome inactivation (XCI) occurs during development, coupled with cell differentiation. In somatic cells, XCI is random, whereas in extraembryonic tissues, XCI is imprinted in that the paternally inherited X chromosome is preferentially inactivated. Inactivation is initiated from an X-linked locus, the X-inactivation center (Xic), and inactivity spreads along the chromosome toward both ends. XCI is established by complex mechanisms, including DNA methylation, heterochromatinization, and late replication. Once established, inactivity is stably maintained in subsequent cell generations. The function of an X-linked regulatory gene, Xist, is critically involved in XCI. The Xist gene maps to the Xic, it is transcribed only from the inactive X chromosome, and the Xist RNA associates with the inactive X chromosome in the nucleus. Investigations with Xist-containing transgenes and with deletions of the Xist gene have shown that the Xist gene is required in cis for XCI. Regulation of XCI is therefore accomplished through regulation of Xist. Transcription of the Xist gene is itself regulated by DNA methylation. Hence, the differential methylation of the Xist gene observed in sperm and eggs and its recognition by protein binding constitute the most likely mechanism regulating imprinted preferential expression of the paternal allele in preimplantation embryos and imprinted paternal XCI in extraembryonic tissues. This article reviews the mechanisms underlying XCI and recent advances elucidating the functions of the Xist gene in mice and humans.

The construction of cDNA libraries from human single preimplantation embryos and their use in the study of gene expression during development.

PURPOSE: The construction and application of polymerase chain reaction (PCR)-based cDNA libraries from unfertilized human oocytes and single preimplantation-stage embryos are described. The purpose of these studies is to provide a readily available resource for the study of gene expression during human preimplantation development. METHODS: Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. We then constructed cDNA libraries from eight unfertilized oocytes and single two-cell, -4-cell, -7-cell, and blastocyst-stage embryos. Differential display PCR using the libraries as template allows the analysis of stage-specific expression of embryonic genes. Genomic libraries are also prepared from parental samples (cumulus cells and sperm) corresponding to the individual embryo generating the cDNA library. RESULTS: The complexities (between 10(5) and 10(6) clones) of the human embryo libraries indicate that they may represent the entire active gene population at these early stages of human development. Nucleotide sequence analyses of random clones showed the presence of a variety of transcripts, such as the human transposable element. LINE-1, Alu repeat sequences, housekeeping genes, and tissue-specific genes, (e.g., alpha-globin, FMR-1, and interleukin-10). Also present at the expected frequency are the ubiquitous cytoskeletal elements, beta-actin, keratin-18, and alpha-tubulin. In addition to cDNAs corresponding to known expressed sequence tags (ESTs) in the GenBank and dbEST databases, a high proportion of novel sequences was also detected. Several cDNAs were detected only at specific stages of preimplantation development by the differential display analysis. CONCLUSIONS: PCR-based cDNA libraries from single human preimplantation embryos provide a new and important resource for the identification and study of novel genes or gene families. As such, they will increase our basic understanding of the molecular control of human development. In the clinical context, the libraries identify the time of onset of specific genes, and hence the diseases resulting from mutation of these genes, and provide information about new methods of preimplantation diagnosis. The molecular analysis of early gene transcription in human embryogenesis may be expected to lead to advances in contraception, assisted reproduction, and preimplantation genetic diagnosis.

Interviewing suspected victims of child maltreatment in the emergency department.

Every day, children suspected of being maltreated present to emergency departments throughout the United States. These children should be given the opportunity to discuss what happened to them. Statements made by an abused child can become powerful evidence in court, and the legality of such statements could be strengthened by the use of forensic interview techniques. The ED interview is not an investigative interview. It should be used to obtain information needed to assess, treat, and report cases of suspected child maltreatment. Ideally, the child should be separated from the caregiver and the setting should provide safety and privacy. The nurse begins the interview with an opened-ended question such as "What happened?" and then progress, if necessary, to more directed for focused questions. Such progression creates confidence in the reliability of the child's responses. Finally, accurate and detailed charting is crucial in a child maltreatment case. The nurse must document the interview using the child's exact words and noting the child's behavior and demeanor at the time the statements were made.

Expression of an Xist promoter-luciferase construct during spermatogenesis and in preimplantation embryos: regulation by DNA methylation.

Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females, a process involving a regulatory gene, Xist (X-inactive specific transcript). Xist maps to the X-inactivation centre and is expressed from the inactive X chromosome in female somatic cells and at the time of X inactivation during spermatogenesis in the male. In female preimplantation embryos, Xist demonstrates imprinting in that the paternal allele inherited from the sperm is preferentially expressed. This preferential paternal Xist expression is correlated with paternal X inactivation in the extraembryonic lineages at the blastocyst stage. We have analysed a 233-bp Xist promoter fragment (nt -220 to +13) for its ability to direct appropriate expression and its regulation by DNA methylation. This minimal promoter sequence directs expression of the luciferase reporter gene following injection of the construct into one-cell embryos. In vitro methylation of the construct before injection represses transcription. In six different transgenic lines, expression of the Xist promoter-luciferase transgene occurs only in the testis of the males (as for the endogenous Xist gene). The testis-specific expression is correlated with hypomethylation of the transgene, although to different extents in different lines. Following paternal transmission, expression of the Xist promoter-luciferase construct in preimplantation embryos is correlated with degree of hypomethylation in the testis and the degree of hypomethylation of the transgene in embryos at the morula stage. It is concluded that the patterns of methylation of the transgene in sperm (and in microinjected transgenes) can regulate the activity of the Xist promoter in the preimplantation embryo and thus support the hypothesis that gametic methylation patterns govern imprinted expression of the endogenous Xist gene in development.