Differential expression and imprinting status of Ins1 and Ins2 genes in extraembryonic tissues of laboratory mice.

There are two functional insulin genes in the mouse genome. The Ins2 gene is imprinted and expressed monoallelically from the paternal allele in the yolk sac. In the present study we have re-examined the imprinting status of Ins1. We found that Ins1 is not expressed in the yolk sac of several laboratory mouse strains. The asynchrony of replication at the wild type locus was significantly lower than at imprinted loci and was more similar to non-imprinted loci. Finally, we have taken the advantage of the Ins1(neo) allele created by homologous recombination to examine the allelic usage at this locus. We observed that the neo gene inserted at the Ins1 locus was expressed from both the paternally and the maternally transmitted allele. Therefore, the Ins1 gene does not share any of the basic properties of imprinted genes. On the basis of these data, we concluded that Ins1 locus is unlikely to be imprinted in common laboratory mice.

Did genomic imprinting and X chromosome inactivation arise from stochastic expression?

Both X chromosome inactivation and autosomal genomic imprinting generate a functional hemizygosity. Here we consider models that explain the evolution of genomic imprinting and X chromosome inactivation from novel perspectives. Specifically, we suggest that random (in)activation events are common in genes and gene clusters with a low probability of transcription. These generate variability that natural selection has acted on to evolve stable monoallelic expression. Possible selection forces might include a need for dosage compensation and the prevention of biallelic silencing where a total switch off would be lethal. Two different mechanisms can accomplish regular monoallelic expression - genomic imprinting and gene counting.

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis.

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production.

Biallelic transcription of Igf2 and H19 in individual cells suggests a post-transcriptional contribution to genomic imprinting.

The H19 and insulin-like growth factor 2 (Igf2) genes in the mouse are models for genomic imprinting during development. The genes are located only 90 kb apart in the same transcriptional orientation [1], but are reciprocally imprinted: Igf2 is paternally expressed while H19 is maternally expressed. It has been suggested that expression of H19 and repression of Igf2 (or the converse) on a given chromosome are mechanistically linked and that the parental imprint operates at the level of transcription [2]. Although expression of Igf2 and H19 is thought to be monoallelic, the data have so far been obtained exclusively by looking at steady-state RNA levels using techniques that reflect the average activity of the genes in a cell population [3] [4]. Here, we have adapted a fluorescent in situ hybridisation (FISH) method to detect nascent RNA molecules of Igf2 and H19 at the initial transcription sites in the nuclei of wild-type mouse embryonic liver cells. Nine different transcription patterns were observed, reflecting a high heterogeneity of transcription at the single-cell level. Our observations suggest that regulation of Igf2 and H19 by parental imprinting is much more complex than previously proposed and acts at both transcriptional and post-transcriptional levels.

The genotype and epigenotype synergize to diversify the spatial pattern of expression of the imprinted H19 gene.

Little is known of how the genetic background effects the phenomenon of genomic imprinting. The H19 gene belongs to a cluster of imprinted genes on human chromosome 11. Here we show that the alternative splicing of a human H19 transcript is genotype-specific. Moreover, this variant transcript, which lacks exon 4, is either not found at all, is widely expressed or is confined to extra-villous cytotrophoblasts in first trimester placenta, depending on a combination of the genotype and the sex of the transmitting parent.

Chromosome methylation patterns during mammalian preimplantation development.

DNA methylation patterns were evaluated during preimplantation mouse development by analyzing the binding of monoclonal antibody to 5-methylcytosine (5-MeC) on metaphase chromosomes. Specific chromosome patterns were observed in each cell stage. A banding pattern predominated in chromosomes at the one-cell stage. Banding was replaced at the two-cell stage by an asymmetrical labeling of the sister chromatids. Then, the proportion of asymmetrical chromosomes decreased by one-half at each cell division until the blastocyst stage, and chromosomes became progressively symmetrical and weakly labeled. Our results indicate that chromosome demethylation is associated with each DNA replication and suggest that a passive mechanism predominates during early development.

Early recognition of pregnancy by the maternal immune system.

PROBLEM: Immunologic recognition of pregnancy is important for normal gestation. Successful pregnancy is characterized by a Th2 dominance, whereas there is a Th1 dominance in failed pregnancies. We assume that a signal given by the fertilized ovum induces a Th2 shift, necessary for a normal outcome. In vitro fertilization provides a tool for testing this possibility. METHOD OF STUDY: Phytohemagglutinin-stimulated lymphocytes were incubated for 48 hr in the presence of culture media from in vitro fertilized eggs, as well as in follicular fluid (FF) and control supernatants. Total RNA was isolated from the lymphocytes by the guanidine-isothiocyanate method and interleukin (IL)-10 mRNA expression was detected by reverse transcription-polymerase chain reaction. RESULTS: Ten percent of the activated lymphocytes incubated with FF expressed IL-10 mRNA, whereas 88% of the lymphocytes activated with supernatants of sperm + oocytes gave a positive signal. Significantly (P < 0.05) fewer (50%) lymphocytes stimulated in the presence of control supernatants also expressed mRNA for IL-10. In supernatants of activated lymphocytes incubated with the culture medium of spermia + oocytes, the concentration of IL-10 was significantly higher than in the lymphocytes incubated with FF. CONCLUSION: These data suggest that the presence of the fertilized ovum induces a Th2 shift, which enables pregnancy to proceed to term.

Imprinting at the mouse Ins2 locus: evidence for cis- and trans-allelic interactions.

The mouse gene encoding preproinsulin 2 (Ins2) is located on the distal end of chromosome 7 in a region of several hundred kilobases that contains several imprinted genes. The exclusive expression of the Ins2 paternal allele in the visceral yolk sac during the last part of gestation indicates that Ins2 also is imprinted. However, in other tissues in which Ins2 is expressed, both alleles are active at all developmental stages. Taking advantage of two mouse strains carrying different null mutations introduced at the Ins2 locus via homologous recombination in ES cells, we examined whether genes inserted at the Ins2 locus become imprinted and have the same restricted pattern of monoallelic expression. In the first null allele, Ins2 was replaced by LacZ, under the control of the endogenous Ins2 promoter, and a Neo cassette with its own promoter was inserted 3' to LacZ (Zneo allele). In the second null allele, Ins2 and its promoter were replaced by the same Neo cassette (Neo allele). Expression of the maternally and paternally inherited genes was monitored by RT-PCR performed on various reciprocal crosses involving the two mutants and the wildtype alleles. In (Zneo x wildtype) F1 embryos, the pattern of LacZ expression was similar to that of Ins2; i.e., LacZ is expressed in the yolk sac only when paternally inherited, while its expression in the embryo proper is independent of its paternal or maternal origin. For both of the mutant alleles, Neo was transcribed only when paternally inherited, in the yolk sac as well as in the embryo. Unexpectedly, we found that LacZ transcription on the maternal chromosome varied depending on the nature of the allele on the paternal chromosome. While fully expressed in the embryo when the paternal chromosome carries the wildtype allele, the maternally inherited LacZ is extinguished when the paternal allele is the Neo allele. The major conclusion from our results is that individual genes introduced into an imprinted chromosomal domain can become imprinted, indicating the influence of long-range cis-acting effects. In addition, our data suggest that the two parental alleles may "communicate" with each other and influence the transcription at the locus.

Imprinted chromosomal regions of the human genome display sex-specific meiotic recombination frequencies.

BACKGROUND: Meiotic recombination events do not occur randomly along a chromosome, but appear to be restricted to specific regions. In addition, some regions in the genome undergo recombination more frequently in the germ cells of one sex than the other. Genomic imprinting, the process by which the two parental alleles of a gene are differentially marked, is another genetic phenomenon associated with inheritance from only one parent or the other. The mechanisms that control meiotic recombination and genomic imprinting are unknown, but both phenomena necessarily depend on the presence of some DNA signal sequences and/or on the structure of the surrounding chromatin domain. RESULTS: In the present study, we compared the frequencies of sex-specific recombination events in three chromosomal regions of the human genome that contain clustered imprinted genes. Alignment of the genetic and physical maps of the ZNF127-SNRPN-IPW-PAR-5-PAR-1 region on chromosome 15q11-q13 (associated with Prader-Willi and Angelman syndromes) and the IGF2-H19 region on chromosome 11p15.5 (associated with Beckwith-Wiedemann syndrome) shows that both regions recombine with very high frequency during male meiosis, and with very low frequency during female meiosis. A third region around the WT-1 gene on chromosome 11p13 also recombines with higher frequency during male meiosis. CONCLUSIONS: The results show that the two best-known imprinted regions in the human genome are characterized by significant differences in recombination frequency during male and female meioses. A third, less well-characterized, imprinted region shows a similar sex-specific bias. On the basis of these observations, we propose a model suggesting that the region-specific differential accessibility of DNA that leads to differential recombination rates during male and female meioses also leads to the male- and female-specific modification of the signal sequences that control genomic imprinting.

Tissue- and developmental stage-specific imprinting of the mouse proinsulin gene, Ins2.

We have investigated the imprinting status of two insulin genes using an interspecific recombinant congenic mouse strain carrying Ins1 and Ins2 alleles from Mus spretus on a C57BL/6 genetic background. At Days 12.5, 13.5, and 14.5 of gestation, expression of both parental alleles of both Ins1 and Ins2 was detected in the bodies of the embryos. In the heads, only Ins2 expression was detected, and, again, both parental alleles were expressed. In yolk sacs, only Ins2 transcripts were found. Both parental alleles were expressed on Day 12.5, but the expression of the maternal allele gradually declined with only the paternal allele remaining active by Day 14.5. Thus, Ins2 is subject to genomic imprinting in the yolk sac. This imprinting is not only tissue-specific, but appears to be a multistep process with postzygotic events likely to play an important role in repression of the maternal allele.

The zonal expression of chicken cartilage matrix protein gene in the developing skeleton of transgenic mice.

Cartilage matrix protein (CMP) is a major noncollagenous glycoprotein of hyaline cartilage with a molecular mass of about 148 kDa. It has been proposed to be involved in matrix organization by its interactions with proteoglycan and type II collagen. The 54-kDa monomers form homotrimers stabilized by disulfide bonds. The gene for chicken cartilage matrix protein was isolated, and its regulation has been studied recently in transient expression experiments. To learn more about the spatial and temporal expression of the gene during ontogenic development, we created transgenic mice via microinjection of a 21.8-kb genomic fragment, encoding the chicken cartilage matrix protein. None of the founder animals exhibited any abnormal phenotype. The developmental stage-specific expression of the transgene was examined by immunostaining with a chicken CMP specific antiserum at different stages of embryonic development in cartilage from different sources: lower and upper limb, vertebrae, ribs and nasal septum. The level of transgene expression showed marked differences in various zones of cartilage. Briefly, high levels were found in the zones of proliferating chondrocytes, while little if any transgene product was detected in the very early and hypertrophic stage of chondrogenesis. The expression pattern of the transgene correlated with the endogenous mouse CMP and did not cause any morphological changes detectable by microscopic analysis of cartilage. These data indicate that the injected CMP gene with its flanking sequences contained all the information necessary for cell type-specific expression in transgenic mice.

Cis effect of lacZ sequences in transgenic mice.

Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving the Escherichia coli beta-galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression in HMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of double HMG-lacZ and HMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that the lacZ sequence might interfere negatively with the expression of the adjacent HMG-cat transgene.

Biopsy and sex determination by PCR of IVF bovine embryos.

Sex of early bovine embryos was determined by polymerase chain reaction (PCR) using a single blastomere removed at the 16-32 cell stage. Embryos were produced in vitro and biopsied on the fifth day after in vitro fertilization. Biopsied embryos were cultured on a cumulus cell monolayer until embryo transfer. For the PCR, one pair of bovine-specific and one pair of Y-chromosome-specific primers were used. Definite signals following PCR amplification were obtained in 95.4% of cases indicating that one blastomere from a preimplantation bovine embryo is sufficient for sex determination by PCR. Nineteen biopsied embryos of predetermined sex were transferred into synchronized recipient females to examine their developmental potential in vivo. Ten of the recipients (52.6%) were found to be pregnant by ultrasonography 25 days after transfer. This result did not differ significantly from that achieved with the use of the control non-manipulated IVF embryos (54.1%; P > 0.1).

Polyclonal origin of pancreatic islets in aggregation mouse chimaeras.

In the present study, we have examined the origin and growth pattern of the beta cells in pancreatic islets, to determine whether a single progenitor cell gave rise to all the precursors of the islets, or if each of a few progenitor cells is the founder of a different islet, or if each islet is a mixture of cells originating from a pool of progenitor cells. Aggregation mouse chimaeras where the pancreatic beta cells derived from each embryo can be identified in the islets on histological sections were analyzed. In two chimaeras, all the islets contained cells from both the aggregated embryo. This clearly demonstrates that each islet resulted from several independent cells. In addition, the beta cells derived from either embryo component were in very small clusters in the islets, suggesting that in situ cell division did not account significantly for islet growth.

Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalasin D.

The microfilament inhibitor cytochalasin D inhibits extrusion of the first polar body when present during the first meiotic division of mouse oocytes; however, it does not interfere with anaphase movement of chromosomes, and thus induces the formation of tetraploid oocytes. After the separation of chromosomes in anaphase, two spindles start to assemble. However, they merge rapidly and a single meiotic spindle forms. During the transition between metaphase I and metaphase II, in the presence of cytochalasin D, a drop in histone kinase activity takes place demonstrating a transitional decrease in the activity of the maturation promoting factor. These oocytes can be activated parthenogenetically a few hours after washing out the inhibitor. After completion of the second meiotic division and extrusion of a polar body, they contain a diploid number of chromosomes. They are genetically identical to each other and to their mother. Such eggs develop to the blastocyst stage and can implant in the uteri of foster mothers. Most of these fetuses die before the 9th day of gestation, as do diploid control fetuses treated with cytochalasin D during the second meiotic division. The heterozygous state of the experimental embryos obtained after activation of eggs recovered from heterozygous females and treated with cytochalasin D during the first meiotic division was confirmed using a glucose-phosphate isomerase assay. This technique allows the production of genetic clones of parthenogenetic embryos by simple means.

Tcp-1 gene is not responsible for the maternal lethality effect of Thp mutation in mice.

Embryos receiving either the Thp or the twLub2 mutation from their mother die during gestation. In contrast, heterozygous embryos receiving the mutant chromosome from the father are completely viable. In both Thp and twLub2 mutants, one chromosome 17 carries a deletion, which suggests the existence of a discrete maternal lethality effect locus (Tme). In the region whose deletion is responsible for the Thp or twLub2 mutation, a single gene has been cloned so far, the Tcp-1 gene. In the present study, we examined the expression of Tcp-1 gene in mutant embryos carrying either a maternal or a paternal Thp chromosome to test whether this gene could be involved in the Tme effect. We found that the levels of Tcp-1-specific transcripts were similar in both mutant embryos from reciprocal crosses and corresponded to half the levels found in the normal littermates. In addition, the paternal and maternal Tcp-1 alleles had identical methylation patterns. These results indicate that Tcp-1 is not responsible for the maternal lethality effect, and therefore is not located at the Tme locus.